Method of identifying inhibitors of DHODH

ABSTRACT

The present invention provides a compound capable of binding to the ubiquinone binding site of DHODH which contains a non-aromatic ring system as a core structure, a group capable of interacting with structural elements of subsite 2 or 3 of the ubiquinone binding site of DHODH and a group capable of interacting hydrophobically with structural elements of subsite 1 of the ubiquinone binding site of DHODH. Furthermore, the present invention provides a compound capable of binding to the ubiquinone binding site of DHODH which contains an aromatic ring system as a core structure, a group capable of interacting with residues His 56 and/or Tyr 356 of subsite 3 of the ubiquinone binding site of DHODH and a group capable of interacting hydrophobically with structural elements of subsite 1 of the ubiquinone binding site of DHODH.

The present invention relates to a polypeptide which comprises theligand binding domain of human dihydroorotate dehydrogenase (DHODH), thecrystalline forms of this polypeptide complexed with newantiproliferative agents and the use of these crystalline forms todetermine the three dimensional structure of the ubiquinone binding siteof DHODH complexed with the ligands. The invention also refers to theuse of the three dimensional structure of the ubiquinone binding site ofDHODH in methods of designing and/or identifying potential inhibitors ofdihydroorotate dehydrogenase (DHODH), for example, compounds which areinhibitors of the ubiquinone binding site, for example, compounds whichinhibit the binding of a native substrate to the ubiquinone binding siteof DHODH.

Inhibitors of DHODH, an enzyme of the pyrimidine biosynthesis, andpharmaceutical compositions containing them, are useful, for example,for the treatment of rheumatoid arthritis (RA). Its treatment with usualmedications as for example non-steroid anti-inflammatory agents is notsatisfactory. In view of the increasing ageing of the population,especially in the developed Western countries or in Japan, thedevelopment of new medications for the treatment of RA is urgentlyrequired.

The DHODH inhibiting leflunomide (ARAVA) [EP 780128, WO 97/34600] is thefirst medicament of this class of compounds (leflunomides) for thetreatment of RA. Leflunomide has immunomodulatorial as well asanti-inflammatorial properties [EP 217206, DE 2524929].

In the body, DHODH catalyzes the synthesis of pyrimidines, which arenecessary for cell growth. An inhibition of DHODH inhibits the growth of(pathologically) fast proliferating cells, whereas cells which grow atnormal speed may obtain their required pyrimidine bases from the normalmetabolic cycle. The most important types of cells for the immuneresponse, the lymphocytes, use exclusively the synthesis of pyrimidinesfor their growth and react particularly sensitively to DHODH inhibition.Substances that inhibit the growth of lymphocytes are importantmedicaments for the treatment of auto-immune diseases.

WO 99/45926 is a further reference that discloses compounds which act asinhibitors of DHODH. A further object of the present invention is toprovide alternative effective agents which can be used for the treatmentof diseases which require the inhibition of DHODH.

In Structure, 2000, Vol. 8, No. 1, pages 25-33, the structure of humanDHODH in complex with the antiproliferative agents brequinar andleflunomide are described.

In Structure, 2000, Vol. 8, No. 1, pages 1227-1238, crystal structuresof DHODH B and its product complex are determined. In PharmaceuticalReasearch, 1998, Vol. 15, No. 2, pages 286-295, and in BiochemicalPharmacology, 1990, Vol. 40, No. 4, pages 709-714, thestructure-activity relationship of leflunomide and quinoline carboxylicacid analogues is analyzed.

In the Journal of Medicinal Chemistry, 1999, Vol. 42, pages 3308-3314,virtual combinatorial syntheses and computational screening of newpotential anti-Herpes compounds are described. In Table 3 on page 3313experimental results regarding IC₅₀ and cytotoxicity are presented for2-(2,3-difluorophenylcarbamoyl)-1-cyclopentene-1-carboxylic acid,2-(2,6-difluorophenylcarbamoyl)-1-cyclopentene-1-carboxylic acid and2-(2,3,4-trifluorophenyl-carbamoyl)-1-cyclopentene-1-carboxylic acid.

SUMMARY OF THE INVENTION

In one embodiment, the present invention relates to a polypeptidecomprising the ligand binding domain of human dihydroorotatedehydrogenase (DHODH), crystalline forms of this polypeptide complexedwith a ligand, and the three dimensional structure of the polypeptide,including the three dimensional structure of the ubiquinone binding siteof DHODH.

In another embodiment, the present invention provides a method ofdetermining the three dimensional structure of a crystalline polypeptidecomprising the ubiquinone binding site of DHODH complexed with theligands. The method comprises the steps of (1) obtaining a crystal ofthe polypeptide comprising the ubiquinone binding site of DHODHcomplexed with a ligand; (2) obtaining x-ray diffraction data for saidcrystal; and (3) solving the crystal structure of said crystal by usingsaid x-ray diffraction data and the atomic coordinates for the DHODHcomplex with the ligand.

The invention further relates to a method of identifying a compoundwhich is a potential inhibitor of DHODH. The method comprises the stepsof (1) obtaining a crystal of the polypeptide comprising the ubiquinonebinding site of DHODH complexed with a ligand; (2) obtaining the atomiccoordinates of the polypeptide in said crystal; (3) using said atomiccoordinates to define the ubiquinone binding site of DHODH complexedwith a ligand; and (4) identifying a compound which fits the ubiquinonebinding site. The method can further include the steps of obtaining orsynthesizing the compound to inhibit at least one biological activity ofDHODH, such as enzymatic activity.

In another embodiment, the method of identifying a potential inhibitorof DHODH comprises the step of determining the ability of one or morefunctional groups and/or moieties of the compound, when present in, orbound to, the ubiquinone binding site of DHODH; to interact with one ormore subsites of the ubiquinone binding site of DHODH. Generally, theubiquinone binding site of DHODH is defined by the atomic coordinates ofa polypeptide comprising the ubiquinone binding site of DHODH. If thecompound is able to interact with a preselected number or set ofsubsites, or has a calculated interaction energy with a desired orpreselected range, the compound is identified as a potential inhibitorof DHODH.

BRIEF DESCRIPTION OF THE INVENTION

FIG. 1 schematically depicts the spatial arrangement of the subsites ofDHODH.

FIG. 2 shows the minimal grid screen used for crystallization trails.

DETAILED DESCRIPTION OF THE INVENTION

The human DHODH enzyme is composed of two domains, namely a largeC-terminal domain (Met78 to C-terminus) and a small N-terminal domain(Met30 to Leu68), connected by an extended loop. The large C-terminaldomain can be described best as an α/β-barrel fold with a central barrelof eight parallel β strands surrounded by eight α helices. The redoxsite, formed by the substrate binding site and the site of the cofactorflavine mononucleotide (FMN), is located on this large C-terminaldomain. The small N-terminal domain, on the other hand, consists of twoα helices, α1 and α2, connected by a short loop. This small N-terminaldomain contains the binding site for the cofactor ubiquinone. Thehelices α1 and α2 span a slot of about 10×20 Å in the so-calledhydrophobic patch, with the short α1-α2 loop at the narrow end of thatslot. The slot forms the entrance to a tunnel that ends at the FMNcavity nearby the α1-α2 loop. This tunnel is narrowing towards theproximal redox site and ends with several charged or polar sidechains(Gln47, His56, Tyr356, Thr360 and Arg136). It is evident that ubiquinonewhich can easily diffuse into the mitochondrial inner membrane uses thistunnel to approach the FMN cofactor for a redox reaction.

The structural knowledge mentioned above can be used to design potentialinhibitors of the human DHODH activity targeting the tunnel mentionedabove and competing with ubiquinone for the ubiquinone binding site.Potential inhibitors were co-crystallized with human DHODH (Met30 toArg396) and the three dimensional structures were solved by proteinX-ray crystallography techniques, ten of the solved structures beingthree dimensional structures of human DHODH (Met30 to Arg396) in complexwith compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. These crystalstructures were solved at atomic resolution and the binding modes of theten compounds were analyzed. The structural formulars of theco-crystallized compounds are given below.

Detailed analysis of the three dimensional structure of the DHODH smallN-terminal domain as well as the three dimensional structure of DHODH incomplex with certain inhibitors designed to target the ubiquinonebinding site revealed the presence of a number of subsites. Each subsiteincludes molecular functional groups or moieties capable of formingstabilizing interactions with complementary functional groups ormoieties of an inhibitor.

The found subsites are characterized below according to the propertiesof functional groups or chemical moieties they are complementary to, orthey can interact with in a stabilizing way, for example, groups ormoieties capable of hydrogen bond formation or groups or moieties withhydrophobic (=lipophilic) character. A hydrogen bond is formed between ahydrogen atom covalently bond to an electronegative element (protondonor or hydrogen bond donor) and a lonely electron pair of a secondelectronegative atom (proton acceptor or hydrogen bond acceptor).Hydrogen bonds typically occur when the hydrogen bond donor and thehydrogen bond acceptor are separated by about 2.5 Å and 3.5 Å.Stabilizing hydrophobic or lipophilic interactions occur if two groupsor moieties with hydrophobic/lipophilic character, for example,aliphatic chains or aromatic systems, are separated by distances closeto their van der Waals radii.

The method of identifying a potential inhibitor of DHODH comprises thestep of determining the ability of one or more functional groups and/ormoieties of the compound, when present in the ubiquinone binding site,to interact with one or more subsites of the ubiquinone binding site.Preferably, the ubiquinone binding site is defined by the atomiccoordinates of a polypeptide comprising the ubiquinone binding site ofDHODH. If the compound is able to interact with a preselected number orset of subsites, the compound is identified as a potential inhibitor ofDHODH.

A functional group or moiety of the compound is said to “interact” witha subsite of the ubiquinone binding site if it participates in anenergetically favourable, or stabilizing, interaction with one or morecomplementary moieties within the subsite.

Two chemical moieties are “complementary” if they are capable, whensuitably positioned, of participating in an attractive, or stabilizing,interaction, such as an electrostatic or an van der Waals interaction.Typically, the attractive interaction is an ion-ion, a salt bridge,ion-dipole, dipole-dipole, hydrogen bond, pi-pi or hydrophobicinteraction. An extreme case of attractive interaction is the formationof a covalent bond by a chemical reaction between the test compound andthe enzyme. For example, a negatively charged moiety and a positivelycharged moiety are complementary because, if suitably positioned, theycan form a salt bridge. Likewise, a hydrogen bond donor and a hydrogenbond acceptor are complementary if suitably positioned.

Preferably, the groups capable of hydrogen bond formation (“HB”) areselected from halogen, such as fluorine, chlorine, bromine and iodine,NO₂, haloalkyl, haloalkyloxy, CN, hydroxyl, amino, hydroxylamine,hydroxamic acid, carbonyl, carbonic acid, sulfonamide, amide, sulfone,sulfonic acid, alkylthio, alkoxy, ester, hydroxyalkylamino group, andother groups including a heteroatom having at least one lone pair ofelectrons, such as groups containing trivalent phosphorous, di- andtetravalent sulfur, oxygen and nitrogen atoms;

Preferably, hydrophobic groups (“H”) are selected from groups, such aslinear, branched or cyclic alkyl groups; linear, branched or cyclicalkenyl groups; linear, branched or cyclic alkynyl groups; aryl groups,such as mono- and polycyclic aromatic hydrocarbyl groups and mono- andpolycyclic heteroaryl groups;

Preferably, negatively charged groups (“N”) are selected from groups,such as carboxylate, sulfonamide, sulfamate, boronate, vanadate,sulfonate, sulfinate and phosphonate groups. A given chemical moiety cancontain one or more of these groups.

In the following a detailed description of identified subsites isprovided. Residue numbering and atom labeling is identical to thenumbering and labeling in Tables 29, 30, and 31.

Subsite 1: Hydrophobic pocket; interacting chemical moieties: H;

Residues involved: Leu 42; Met 43; Leu 46; Ala 55; Ala 59; Phe 98; Met111; Leu 359; Pro 364;

Non-hydrogen atoms which interact with H: Leu 42 CB, CG, CD1, CD2; Met43 SD, CE; Leu 46 CB, CG, CD1, CD2; Ala 55 CB; Ala 59 CA, CB; Phe 98 CG,CD1, CD2, CE1, CE2; Met 111 SD, CE; Leu 359 CA, CB, CG, CD1, CD2; Pro364 CB, CD, CG;

Preferably for the hydrophobic interacting with subsite 1, the group isselected from aryl groups, such as an aromatic group having five tofifteen carbon atoms, which can optionally be substituted by one or moresubstituents R′. More preferably the aryl group is a phenyl group, suchas —CH₂Ph, —C₂H₄Ph, —CH═CH-Ph, —C≡C-Ph, -o-C₆H₄—R′, -m-C₆H₄—R′,—P—C₆H₄—R′, —CH₂—C₆H₄—R′, -m-CH₂—C₆H₄—R′, -p-CH₂—C₆H₄—R′; or a biphenylgroup, in which the phenyl rings can optionally be substituted by one ormore substituents R′, such biphenyl groups are —C₆H₄—C₆H₅;—C₆H₄—C₆H₄—R′; —C₆H₃—R′—C₆H₅; —C₆H₃—R′—C₆H₄—R′;

—C₆H₃—R′—C₆H₄—R′; —C₆H₄—O—C₆H₅; —C₆H₃—R′—O—C₆H₄—R′; —C₆H₄—O—C₆H₄—R′;—C₆H₃—R′—O—C₆H₅; —C₆H₄—O—CH₂—C₆H₅; —C₆H₃—R′—O—CH₂—C₆H₄—R′;—C₆H₄—O—CH₂—C₆H₄—R′; —C₆H₃—R′—O—CH₂—C₆H₅;

R′ is independently H, —CO₂R″, —CONHR″, —CR″O, —SO₂NR″,—NR″—CO-haloalkyl, —NO₂, —NR″—SO₂-haloalkyl, —NR″—SO₂-alkyl, —SO₂-alkyl,—NR″—CO-alkyl, —CN, alkyl, cycloalkyl, aminoalkyl, alkylamino, alkoxy,—OH, —SH, alkylthio, hydroxyalkyl, hydroxyalkylamino, halogen,haloalkyl, haloalkyloxy, aryl, arylalkyl or heteroaryl;

R″ is independently hydrogen, haloalkyl, hydroxyalkyl, alkyl,cycloalkyl, aryl, heteroaryl or aminoalkyl;

R′ is preferably F, Cl, Br, I, CF₃, OCF₃, or OCH₃;

Subsite 2: First anion binding site; interacting with HB, N, HB and N,HB and HB, or N and N;

Residues involved: Gln 47; Arg 136; one conserved water molecule

Non-hydrogen atoms which interact with HB and N: Glu 47 OE1, NE2; Arg136 NE, NH1, NH2; conserved water molecule OH2.

preferably for one or two hydrogen bond formations with subsite 2 thegroup is selected from halogen, such as fluorine, chlorine, bromine andiodine, NO₂, haloalkyl, haloalkyloxy, CN, hydroxyl, amino,hydroxylamine, hydroxamic acids, carbonyl, carbonic acid, sulfonamide,amide, sulfone, sulfonic acid, alkylthio, alkoxy, such as methoxy,ester, hydroxyalkylamino, carboxylate, tetrazole, sulfonamide,sulfamate, boronate, vanadate, sulfonate, sulfinate and phosphonategroup, more preferably from a carboxylate, sulfonamide, sulfamate,sulfonate, carbonyl or carbonic acid group.

Subsite 3: Second anion binding site; interacting with HB, N, HB and N,HB and HB, or N and N;

Residues involved: His 56; Tyr 356; Tyr 147 (interacting via a conservedwater molecule);

Non-hydrogen atoms which interact with HB and N: His 56 N, ND1; Tyr 356OH; Tyr 147 OH (interacting via a conserved water molecule);

preferably for one or two hydrogen bond formations with subsite 2 thegroup is selected from halogen, such as fluorine, chlorine, bromine andiodine, NO₂, haloalkyl, haloalkyloxy, CN, hydroxyl, amino,hydroxylamine, hydroxamic acids, carbonyl, carbonic acid, sulfonamide,amide, sulfone, sulfonic acid, alkylthio, alkoxy, such as methoxy,ester, hydroxyalkylamino, carboxylate, tetrazole, sulfonamide,sulfamate, boronate, vanadate, sulfonate, sulfinate and phosphonategroup, more preferably from a carboxylate, sulfonamide, sulfamate,sulfonate, carbonyl or carbonic acid group.

Subsite 4: Remote hydrophobic pocket; interacting chemical moieties: H;

Residues involved: Pro 52; Val 134; Arg 136; Val 143; Thr 360; FMN;

Non-hydrogen atoms which interact with H: Pro 52 CB, CG, CD; Val 134 CB,CG1, CG2; Val 143 CB, CG1, CG2; Thr 360 CG2; FMN C7M, C8M;

Preferably for the hydrophobic interacting with subsite 4, the group isselected from such as linear, branched or cyclic C₁-C₆-alkyl groups;such as methyl, ethyl, propyl, butyl, tert. butyl, linear, branched orcyclic C₁-C₆-alkenyl groups; linear, branched or cyclic C₁-C₆-alkynylgroups; aryl groups, such as mono- and bi aromatic hydrocarbyl groups,such as —CH₂Ph, —C₂H₄Ph, —CH═CH-Ph, —C≡C-Ph, -o-C₆H₄—R′, -m-C₆H₄—R,—P—C₆H₄—R, -o-CH₂—C₆H₄—R, -m-CH₂—C₆H₄—R, -p-CH₂—C₆H₄—R and mono- andbicyclic heteroaryl groups, such as thiazol-2-yl, thiazol-4-yl,thiazol-5-yl, isothiazol-3-yl, isothiazol-4-yl, isothiazol-5-yl,1,2,4-oxadiazol-3-yl, 1,2,4-oxadiazol-5-yl, 1,2,4-thiadiazol-3-yl,1,2,4-thiadiazol-5-yl, 1,2,5-oxadiazol-3-yl, 1,2,5-oxadiazol-4-yl,1,2,5-thiadiazol-3-yl, 1-imidazolyl, 2-imidazolyl,1,2,5-thiadiazol-4-yl, 4-imidazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl,4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl,4-pyridazinyl, 2-pyrazinyl, 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl,indolyl, indolinyl, tetrazolyl, benzo-[b]-furanyl, benzo[b]thiophenyl,benzimidazolyl, benzothiazolyl, quinazolinyl, quinoxazolinyl, orpreferably isoxazol-3-yl, isoxazol-4-yl, isoxazol-5-yl, quinolinyl,tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl; all thisgroups can optionally be substituted by one or more substituents R, suchas H, amino, alkoxy, OH, SH, alkylthio, hydroxyalkyl, haloalkyl,haloalkyloxy hydroxyalkylamino, halogen; R is preferably F, Cl, Br, I,CF₃, OCF₃, or OCH₃;

Core: chemical moiety connecting the different moieties interacting withSubsite 1, Subsite 2, Subsite 3, and Subsite 4;

Preferably, the core is selected from cyclic alkyl groups; cyclicalkenyl groups; cyclic alkynyl groups; aryl groups, such as mono- andpolycyclic aromatic hydrocarbyl groups and mono- and polycyclicheteroaryl groups; more preferably it is selected from mono-, orbicyclic aromatic or non-aromatic ring systems, most preferably from5-membered mono-, or bicyclic aromatic or non-aromatic ring systems,such as trans-cyclopentan-1,2-diyl, trans-cyclohexan-1,2-diyl,cis-cyclopentan-1,2-diyl, cis-cyclohexan-1,2-diyl,1-cyclopenten-1,2-diyl, 2-cyclopenten-1,2-diyl, 3-cyclopenten-1,2-diyl,4-cyclopenten-1,2-diyl, 5-cyclopenten-1,2-diyl, 1-cyclopenten-1,3-diyl,1-cyclopenten-1,4-diyl, 1-cyclohexen-1,2-diyl, 1-cyclohepten-1,2-diyl or1-cycloocten-1,2-diyl, 2,5-dihydrothiophene-3,4-diyl,2,5-dihydro-furan-3,4-diyl, 2,5-dihydro-1H-pyrrole-3,4-diyl,2,5-dihydro-1-methyl-pyrrole-3,4-diyl,2,5-dihydro-1-ethyl-pyrrole-3,4-diyl,2,5-dihydro-1-acetyl-pyrrole-3,4-diyl,2,5-dihydro-1-methylsulfonyl-pyrrole-3,4-diyl, thiazol-2-yl,thiazol-4-yl, thiazol-5-yl, isothiazol-3-yl, isothiazol-4-yl,isothiazol-5-yl, 1,2,4-oxadiazol-3-yl, 1,2,4-oxadiazol-5-yl,1,2,4-thiadiazol-3-yl, 1,2,4-thiadiazol-5-yl, 1,2,5-oxadiazol-3-yl,1,2,5-oxadiazol-4-yl, 1,2,5-thiadiazol-3-yl, 1-imidazolyl, 2-imidazolyl,1,2,5-thiadiazol-4-yl, 4-imidazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl,4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl,4-pyridazinyl, 2-pyrazinyl, 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl,indolyl, indolinyl, tetrazolyl, benzo-[b]-furanyl, benzo[b]thiophenyl,benzimidazolyl, benzothiazolyl, quinazolinyl, quinoxazolinyl, orpreferably quinolinyl, tetrahydro-quinolinyl, isoquinolinyl,tetrahydroisoquinolinyl or from a group comprising of:

Bridge: chemical moiety connecting the core with Subsite 1;Preferably, the bridge is selected from —NH; —O; —CO—NH; —NH—CO;—NH—CO—NH; alkyl; —O—CH₂; —CH₂—O; —O—CH₂—CH₂; —CH₂—CH₂—O; —NH—CH₂;—CH₂—NH; —NH—CH₂—CH₂; —CH₂—CH₂—NH; —CH₂—CO—NH; —CH₂—NH—CO;Subsite 5: Solvent anchor; interacting chemical moieties: HBResidues involved: Met 30; Tyr 38; Leu 67;Non-hydrogen atoms which interact with HB: Met 30 O, SD, CE; Tyr 38 OH,CE2, CD2; Leu 67 O;preferably for the hydrogen bond formation with subsite 5, the group isselected from F, Cl, Br, I, CF₃, OCF₃, or OCH₃Subsite 6: Solvent anchor; interacting chemical moieties: H;Residues involved: Leu 68;Non-hydrogen atoms which interact with H: Leu 68 CB, CG, CD1, CD2;Preferably for the hydrophobic interacting with subsite 6, the group isselected from such as linear, branched or cyclic C₁-C₆-alkyl groups;such as methyl, ethyl, propyl, butyl, tert. butyl, linear, branched orcyclic C₁-C₆-alkenyl groups; linear, branched or cyclic C₁-C₆-alkynylgroups; aryl groups, such as mono- and bi aromatic hydrocarbyl groups,such as —CH₂Ph, —C₂H₄Ph, —CH═CH-Ph, —C≡C-Ph, -o-C₆H₄—R′, -m-C₆H₄—R,-p-C₆H₄—R, -o-CH₂—C₆H₄—R, -m-CH₂—C₆H₄—R, -p-CH₂—C₆H₄—R and mono- andbicyclic heteroaryl groups.An alkyl group, if not stated otherwise, denotes a linear or branchedC₁-C₆-alkyl, preferably a linear or branched chain of one to five carbonatoms, a linear or branched C₁-C₆-alkenyl or a linear or branchedC₁₋₆-alkinyl group, which can optionally be substituted by one or moresubstituents R′, preferably by halogen;the C₁-C₆-alkyl, C₁-C₆-alkenyl and C₁₋₆-alkinyl residue may be selectedfrom the group comprising —CH₃, —C₂H₅, —CH═CH₂, —C≡CH, —C₃H₇, —CH(CH₃)₂,—CH₂—CH═CH₂, —C(CH₃)═CH₂, —CH═CH—CH₃, —C≡C—CH₃, —CH₂—C≡CH, —C₄H₉,—CH₂—CH(CH₃)₂, —CH(CH₃)—C₂H₅, —C(CH₃)₃, —C₅H₁₁, —C₆H₁₃, —C(R′)₃,—C₂(R′)₅, —CH₂—C(R′)₃, —C₃(R′)₇, —C₂H₄—C(R′)₃, —C₂H₄—CH═CH₂,—CH═CH—C₂H₅, —CH═C(CH₃)₂, —CH₂—CH═CH—CH₃, —CH═CH—CH═CH₂, —C₂H₄—C≡CH,—C≡C₂H₅, —CH₂—C≡C—CH₃, —C≡C—CH═CH₂, —CH═CH—C≡CH, —C≡C—C≡CH,—C₂H₄—CH(CH₃)₂, —CH(CH₃)—C₃H₇, —CH₂—CH(CH₃)—C₂H₅, —CH(CH₃)—CH(CH₃)₂,—C(CH₃)₂—C₂H₅, —CH₂—C(CH₃)₃, —C₃H₆—CH═CH₂, —CH═CH—C₃H₇, —C₂H₄—CH═CH—CH₃,—CH₂—CH═CH—C₂H₅, —CH₂—CH═CH—CH═CH₂, —CH═CH—CH═CH—CH₃, —CH═CH—CH₂—CH═CH₂,—C(CH₃)═CH—CH═CH₂, —CH═C(CH₃)CH═CH₂, —CH═CH—C(CH₃)═CH₂, —CH₂—CH═C(CH₃)₂,C(CH₃)═C(CH₃)₂, —C₃H₆—C≡CH, —C≡C—C₃H₇, —C₂H₄—C≡CH₃, —CH₂—C≡C—C₂H₅,—CH₂—C═C—CH═CH₂, —CH₂—CH═CH—C—CH, —CH₂—C≡C—C≡CH, —C≡C—CH═CH—CH₃,—CH═CH—C═C—CH₃, —C≡C—C≡C—CH₃, —C≡C—CH₂—CH═CH₂, —CH═CH—CH₂—C≡CH,—C≡C—CH₂—C≡CH, —C(CH₃)═CH—CH═CH₂, —CH═C(CH₃)CH═CH₂, —CH═CH—C(CH₃)═CH₂,—C(CH₃)═CH—C≡CH, —CH═C(CH₃)—C≡CH, —C≡C—C(CH₃)═CH₂, —C₃H₆—CH(CH₃)₂,—C₂H₄—CH(CH₃)—C₂H₅, —CH(CH₃)—C₄H₉, —CH₂—CH(CH₃)—C₃H₇,—CH(CH₃)—CH₂—CH(CH₃)₂, —CH(CH₃)—CH(CH₃)—C₂H₅, —CH₂—CH(CH₃)—CH(CH₃)₂,—CH₂—C(CH₃)₂—C₂H₅, —C(CH₃)₂—C₃H₇, —C(CH₃)₂—CH(CH₃)₂, —C₂H₄—C(CH₃)₃,—CH(CH₃)—C(CH₃)₃, —C₄H₈—CH═CH₂, —CH═CH—C₄H₉, —C₃H₆—CH═CH—CH₃,—CH₂—CH═CH—C₃H₇, —C₂H₄—CH═CH—C₂H₅, CH₂—C(CH₃)═C(CH₃)₂, —C₂H₄—CH═C(CH₃)₂,—C₄H₈—C≡CH, —C≡C—C₄H₉, —C₃H₆—C═C—CH₃, —CH₂—C≡C—C₃H₇, —C₂H₄—C≡C—C₂H₅;R′ is independently H, —CO₂R″, —CONHR″, —CR″O, —SO₂NR″,—NR″—CO-haloalkyl, —NO₂, —NR″—SO₂-haloalkyl, —NR″—SO₂-alkyl, —SO₂-alkyl,—NR″—CO-alkyl, —CN, alkyl, cycloalkyl, aminoalkyl, alkylamino, alkoxy,—OH, —SH, alkylthio, hydroxyalkyl, hydroxyalkylamino, halogen,haloalkyl, haloalkyloxy, aryl, arylalkyl or heteroaryl;R″ is independently hydrogen, haloalkyl, hydroxyalkyl, alkyl,cycloalkyl, aryl, heteroaryl or aminoalkyl;a cycloalkyl group denotes a non-aromatic ring system containing four toeight carbon atoms, preferably four to eight carbon atoms, wherein oneor more of the carbon atoms in the ring can be substituted by a group X,X being as defined above; the C₄-C₈-cycloalkyl residue may be selectedfrom the group comprising -cyclo-C₄H₇, -cyclo-C₅H₉, -cyclo-C₆H₁₁,-cyclo-C₇H₁₃, -cyclo-C₈H₁₅;an alkoxy group denotes an O-alkyl group, the alkyl group being asdefined above; the alkoxy group is preferably a methoxy, ethoxy,isopropoxy, t-butoxy or pentoxy group;an alkylthio group denotes an S-alkyl group, the alkyl group being asdefined above.an haloalkyl group denotes an alkyl group which is substituted by one tofive halogen atoms, the alkyl group being as defined above; thehaloalkyl group is preferably a —C(R¹⁰)₃, —CR¹⁰(R^(10′))₂,—CR¹⁰(R^(10′))R^(10″), —C₂(R¹⁰)₅, —CH₂—C(R¹⁰)₃, —CH₂—CR¹⁰(R^(10′))₂,—CH₂—CR¹⁰(R^(10′))R^(10″), —C₃(R¹⁰)₇ or —C₂H₄—C(R¹⁰)₃, wherein R¹⁰,R^(10′), R^(10″) represent F, Cl, Br or I, preferably F;a hydroxyalkyl group denotes an HO-alkyl group, the alkyl group being asdefined above;an haloalkyloxy group denotes an alkoxy group which is substituted byone to five halogen atoms, the alkyl group being as defined above; thehaloalkyloxy group is preferably a —OC(R¹⁰)₃, —OCR¹⁰(R^(10′))₂,—OCR¹⁰(R^(10′))R^(10″), —OC₂(R¹⁰)₅, —OCH₂—C(R¹⁰)₃, —OCH₂—CR¹⁰(R^(10′))₂,—OCH₂—CR¹⁰(R^(10′))R^(10″), —OC₃(R¹⁰)₇ or —OC₂H₄—C(R¹⁰)₃, wherein R¹⁰,R^(10′), R^(10″) represent F, Cl, Br or I, preferably F;a hydroxyalkylamino group denotes an (HO-alkyl)₂-N— group orHO-alkyl-NH— group, the alkyl group being as defined above;an alkylamino group denotes an HN-alkyl or N-dialkyl group, the alkylgroup being as defined above;a halogen group is chlorine, bromine, fluorine or iodine, fluorine beingpreferred;an aryl group preferably denotes an aromatic group having five tofifteen carbon atoms, which can optionally be substituted by one or moresubstituents R′, where R′ is as defined above; the aryl group ispreferably a phenyl group, —CH₂Ph, —C₂H₄Ph, —CH═CH-Ph, —C—C-Ph,-o-C₆H₄—R′, -m-C₆H₄—R′, -p-C₆H₄—R′, -m-CH₂—C₆H₄—R′, -m-CH₂—C₆H₄—R′,-p-CH₂—C₆H₄—R′;a heteroaryl group denotes a 5- or 6-membered heterocyclic group whichcontains at least one heteroatom like O, N, S. This heterocyclic groupcan be fused to another ring. For example, this group can be selectedfrom a thiazol-2-yl, thiazol-4-yl, thiazol-5-yl, isothiazol-3-yl,isothiazol-4-yl, isothiazol-5-yl, 1,2,4-oxadiazol-3-yl,1,2,4-oxadiazol-5-yl, 1,2,4-thiadiazol-3-yl, 1,2,4-thiadiazol-5-yl,1,2,5-oxadiazol-3-yl, 1,2,5-oxadiazol-4-yl, 1,2,5-thiadiazol-3-yl,1-imidazolyl, 2-imidazolyl, 1,2,5-thiadiazol-4-yl, 4-imidazolyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-furanyl, 3-furanyl, 2-thienyl,3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl, 4-pyridazinyl, 2-pyrazinyl,1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl, 1H-tetrazol-2-yl,1H-tetrazol-3-yl, tetrazolyl, indolyl, indolinyl, benzo-[b]-furanyl,benzo[b]thiophenyl, benzimidazolyl, benzothiazolyl, quinazolinyl,quinoxazolinyl, or preferably quinolinyl, tetrahydroquinolinyl,isoquinolinyl, tetrahydroisoquinolinyl group. This heterocyclic groupcan optionally be substituted by one or more substituents R′, where R′is as defined above. In another embodiment, the present inventionprovides DHODH inhibitors, and methods of use thereof, which are capableof binding to the ubiquinone binding site of DHODH, for example,compounds wich are identified as inhibitors of DHODH or which aredesigned by the methods described above to inhibit DHODH. For example,the invention includes compounds which interact with one or more,preferably two or more, and more preferably, three or more of DHODHsubsites 1 to 6.

Preferably an inhibitor of DHODH should have a core-unit and interactwith subsite 1, 2, 3 and 5 or an inhibitor of DHODH should have acore-unit and interact with subsite 1, 2 and 5, or an inhibitor of DHODHshould have a core-unit and interact with subsite 1, 3 and 5.

More preferably an inhibitor of DHODH should have a core-unit andinteract with subsite 1, 2 and 3, or an inhibitor of DHODH should have acore-unit and interact with subsite 1 and 3. In FIG. 1, the spatialarrangement of the subsites is depicted schematically.

The three dimensional structure published by Shenpig et al. shows humanDHODH(Met30-Arg396) in complex with brequinar and the leflunomidemetabolite A771726, respectively. The main interaction in the binding ofbrequinar to DHODH is the formation of a salt bridge between the carboxygroup of brequinar and the sidechain of Arg136. In particular, the saltbridge is formed between the carboxylic group and the atoms NE, NH1 orNH2. More precisely, the above mentioned subsite 2, the first anionbinding site, is addressed in this kind of interaction. In thefollowing, this type of interactioned will be termed “brequinar-likebinding mode”.

Analysis of the three dimensional structures of human DHODH in complexwith ligands presented here clearly shows a new binding mode forinhibitors containing a carboxylic acid group. This binding mode differsfrom the brequinar-like binding mode in interacting not with subsite 2but with subsite 3, termed the second anion binding site. In particularthis is true for inhibitor compounds 1, 4, 5, 7 and 8 as can be seenfrom Table 29. This so far unobserved binding mode will be termed“non-brequinar-like” binding mode in the following.

The “non-brequinar-like” binding mode is characterized by a number ofhydrogen bonds formed between the ligand and protein residues belongingto subsite 3. In particular this residues are His 56, Tyr 356 and Tyr147. Non-hydrogen atoms involved in the formation of hydrogen bonds areN and ND1 of His 56, the oxygen of the hydroxyl group of Tyr 356 and theoxygen of the hydroxyl group of Tyr 147. The latter interaction involvesa conserved water molecule bridging the space between the carboxylfunction of the ligand molecule and the hydroxyl group of the tyrosineresidue 147.

Similar findings can be seen in the three dimensional structure of humanDHODH in complex with the compounds 2, 6 and 10. As can be seen clearlyfrom the electron density map, the compounds 2, 6 and 10 are able toutilize both anion binding sites (subsite 2 and 3) by adopting twoalternative conformations. Therefore, both a brequinar-like and anon-brequinar-like binding mode can be utilized. In the brequinar-likebinding mode the carboxy group of compounds 2, 6 and 10 forms hydrogenbonds to the sidechains of residues Gln 47 and Arg 136. In thenon-brequinar-like binding mode the five membered ring of compounds 2, 6and 10 containing the carboxy group is rotated by almost 180 degrees andforms hydrogen bonds to residues His 56 and Tyr 356. Non-hydrogen atomsinvolved in the formation of hydrogen bonds are N and ND1 of His 56 andthe oxygen of the hydroxyl group of Tyr 356.

The compounds 2, 3 and 4 are particularly interesting for astructure-activity-relationship (SAR) analysis. These molecules differonly in the degree of ring substitution (see structures above). Clearly,one can observe a correlation between the number of fluorinatedpositions at the aromatic ring in the middle of the molecules and thecorresponding IC₅₀ values. The higher the number of ring substituentsthe lower the IC₅₀. Interestingly compound 2 and compound 3 display boththe brequinar-like and non-brequinar-like binding mode in the crystalstructure (see table 27). It is quite reasonable to speculate whetherthe ring substituents exhibit a steering effect on the five memberedring and by such facilitate the formation of the more favourablebrequinar-like binding mode. Therefore, the presence of both bindingmodes might explain the increased affinity of this compounds. TABLE 27Relation of inhibitor binding mode and degree of ring substitutions.Structures of the compounds are shown above. Compound Brequinar-likeNon-Brequinar-like 3 X X 2 X X 4 X

A similar structure-activity-relationship can be deduced from thecrystal structures of humann DHODH in complex with compounds 9 and 10.These compounds carry a sulfur atom at an ortho position with respect tothe carboxylic group in the five membered ring. Compound 10 is singlesubstituted with fluorine at the biaryl ring system, whereas compound 9bears two substituents. Interestingly, compound 9 exhibits a purebrequinar-like binding mode whereas compound 10 shows both alternatives.Additionally, the sulfur atom in the ortho position on the five memberedring can favourably interact with the protein's subsite 4 (remotehydrophobic pocket). The activity data correlate to a very high degreewith the presence of a particular binding mode (Table 28). Obviously,not only the degree of ring subsitution but also ring planarity mightcontribute to the formation of a particular binding mode. TABLE 28Relation of inhibitor binding mode and degree of ring substitutions.Structures of the compounds are shown above. Compound Brequinar-likeNon-Brequinar-like 9 X 10 X X

From the discussion above several possibilities for further synthesis ofcompounds emerge. First, one could try to stabilize the Brequinar-likeconformation by a more elaborate variation of substitution patterns atthe aromatic ring system. A second way to improve on the affinity mightcomprise the addition of a second functional group, which is able toform hydrogen bonds or salt bridges to the five membered ring oppositeto the position of the carboxy group. Thus the molecule should be ableto address both anion subsites and utilize brequinar-like as well asnon-brequinar-like binding modes at the same time. This is highlysupported by the evidence of structural data. Mobility at the site ofGln47 and Arg136 indicates that the protein should be able to exhibitsufficient conformational flexibility to adopt ligand moleculesdisplaying more demanding sterical requirements.

Another interesting finding is that the DHODH binding pocket is able toselectively discriminate between enantiomeres. Compounds 5 and 6 weresynthesized as a racemic mixtures caused by the presence of a stereocentre at the five membered ring (see above). The racemic mixtures wereused for crystallization experiments. In both cases the refinedstructures unequivocally showed the inhibitor bound in its R-form. It isnot possible to fit the S-enantiomer into the electron density.

The invention further provides a method of designing a compound which isa potential inhibitor of DHODH. The method includes the steps of (1)identifying one or more functional groups capable of interacting withone or more subsites of the ubiquinone binding site of DHODH; and (2)identifying a scaffold which presents the functional group or functionalgroups identified in step 1 in a suitable orientation for interactingwith one or more subsites of the ubiquinone binding site of DHODH. Thecompound which results from attachment of the identified functionalgroups or moieties to the identified scaffold is a potential inhibitorof DHODH. The DHODH ubiquinone binding site is, generally, defined bythe atomic coordinates of a polypeptide comprising the DHODH ubiquinonebinding site.

The present invention also provides several advantages. For example, theinvention provides a new three dimensional structure of a crystallinepolypeptide comprising the ubiquinone binding site of DHODH complexedwith the ligands. This structure enables the rational development ofinhibitors of DHODH by permitting the design and/or identification ofmolecular structures having features which facilitate binding to theubiquinone binding site of DHODH. The methods of use of this structuredisclosed herein, thus, permit more rapid discovery of compounds whichare potentially useful for the treatment of conditions which aremediated, at least in part, by DHODH activity.

The polypeptide preferably comprises the ubiquinone binding site of amammalian DHODH. More preferably the polypeptide comprises theubiquinone binding site of human DHODH. In a preferred embodiment, thepolypeptide is a polypeptide of the present invention, as describedabove.

The polypeptide can be crystallized using methods known in the art, suchas the methods described in Structure, 2000, Vol. 8, No. 1, pages 25-33,to afford polypeptide crystals which are suitable for x-ray diffractionstudies. A crystalline polypeptide/ligand complex can be produced byco-crystallizing the polypeptide with a solution including the ligand.

The atomic coordinates of the polypeptide and the ligand can bedetermined, for example, by x-ray crystallography using methods known inthe art. The data obtained from the crystallography can be used togenerate atomic coordinates, for example, of the polypeptide and ligand,if present. As is known in the art, solution and refinement of the x-raycrystal structure can result in the determination of coordinates forsome or all of the non-hydrogen atoms.

The atomic coordinates of the polypeptide can be used, as is known inthe art, to generate a three-dimensional structure of the ubiquinonebinding site of DHODH. This structure can then be used to assess theability of any given compound, preferably using computer-based methods,to fit into the ubiquinone binding site.

The atomic coordinates of the polypeptide/ligand complex can be used, asis known in the art, to generate a three-dimensional structure of theligand in its binding conformation. This structure can then be used toassess the ability of any given compound, preferably usingcomputer-based methods, to exhibit a similar spatial orientation andelectrostatic and/or van der Waals interactions as the ligand andtherefore, to fit into the addressed binding site.

A compound fits into the ubiquinone binding site if it is of suitablesize and shape to physically reside in the ubiquinone binding site, thatis if it has a shape which is complementary to the ubiquinone bindingsite and can reside in the ubiquinone binding site without significantunfavorable sterical or van der Waals interactions. Preferably, thecompound includes one or more functional groups and/or moieties whichinteract with one or more subsites within the ubiquinone binding site.Computational methods for evaluating the ability of a compound to fitinto the ubiquinone binding site, as defined by the atomic coordinatesof the polypeptide, are known in the art, and representative examplesare provided below.

In another embodiment, the method of identifying a potential inhibitorof DHODH comprises the step of determining the ability of one or morefunctional groups and/or moieties of the compound, when present in theDHODH ubiquinone binding site, to interact with one or more subsites ofthe DHODH ubiquinone binding site. Preferably, the DHODH ubiquinonebinding site is defined by the atomic coordinates of a polypeptidecomprising the DHODH ubiquinone binding site. If the compound is able tointeract with a preselected number of subsites, the compound isidentified as a potential inhibitor of DHODH.

In yet another embodiment, the method of identifying a potentialinhibitor of DHODH comprises the steps of (1) identifying the size andshape of the ligand co-crystallized in the polypeptide/ligand complexand/or identifying functional groups or moieties of the ligand which arecapable to form stabilizing interactions with the polypeptide, and (2)by comparison with these, identifying one or more functional groupsand/or moieties of any given compound which have similar size and shapeas the cocrystallized ligand and/or are capable to form one or moreinteractions to the polypeptide in a similar manner as theco-crystallized ligand. If a compound exhibits one or more of thesefeatures, the compound is identified as a potential inhibitor of DHODH.

A functional group or moiety of the compound is said to “interact” witha subsite of the DHODH ubiquinone binding site if it participates in anenergetically favourable, or stabilizing, interaction with one or morecomplementary moieties within the subsite, as defined above.

A functional group or moiety of the compound is said to interact in a“similar” manner as the co-crystallized ligand if one or more,preferably two or more of its functional groups or moieties capable offorming the attractive interactions mentioned above can be superimposedon those functional groups or moieties of the co-crystallized ligandcapable of forming the attractive interactions. The superposition can beperformed based on the identity of atoms, and/or the identity orsimilarity of functional groups, and/or the similarity of molecularshape and/or the identity or similarity of interaction possibilities.For example, an —OH group of a compound and an —NH group of thecocrystallized ligand may interact in the same way, namely as hydrogenbond donors, with a hydrogen bond acceptor atom suitably positioned inthe enzyme. Therefore, the —OH group and the —NH group are said to havesimilar interaction properties, and a molecule containing an —OH groupmay be superimposed onto a molecule carrying an —NH group at thecorresponding position.

Typically, the assessment of interactions between (1) the test compoundand the DHODH ubiquinone binding site and (2) the superposition of atest compound and the co-crystallized ligand employ computer-basedcomputational methods, such as those known in the art, in which, for thefirst case, possible interactions of a compound with the protein, asdefined by atomic coordinates, are evaluated with respect to interactionstrength by calculating the interaction energy upon binding the compoundto the protein. For the second case, the superposition of a testcompound and the cocrystallized ligand is performed according to theidentity of atoms, and/or the identity or similarity of functionalgroups, and/or the similarity of molecular shape and/or the identity orsimilarity of interaction possibilities in a process termed alignment.Matching atoms/functional groups/shape/interaction possibilities areevaluated and summarized to an alignment score enabling the ranking ofthe tested molecules.

Compounds which have calculated interaction energies within apreselected range or which otherwise, in the opinion of thecomputational chemist employing the method, have the greatest potentialas DHODH inhibitors, can then be provided, for example, from a compoundlibrary or via synthesis, and assayed for the ability to inhibit DHODH.The interaction energy for a given compound generally depends upon theability of the compound to interact with one or more subsites within theprotein catalytic domain.

In one embodiment, the atomic coordinates used in the method are theatomic coordinates set forth in Tables 29, 30, and 31. It is to beunderstood that the coordinates set forth in Tables 29, 30, and 31 canbe transformed, for example, into a different coordinate system, in waysknown to those of skill in the art without substantially changing thethree dimensional structure represented thereby.

In certain cases a moiety of the compound can interact with a subsitevia two or more individual interactions. A moiety of the compound and asubsite can interact if they have complementary properties and arepositioned in sufficient proximity and in a suitable orientation for astabilizing interaction to occur. The possible range of distances forthe moiety of the compound and the subsite depends upon the distancedependence of the interaction, as known in the art. For example, ahydrogen bond typically occurs when a hydrogen bond donor atom, whichbears a hydrogen atom, and a hydrogen bond acceptor atom are separatedby about 2.5 Å and about 3.5 Å. Hydrogen bonds are well known in theart. Generally, the overall interaction, or binding, between thecompound and the ubiquinone binding site will depend upon the number andstrength of these individual interactions.

The ability of a test compound to interact with one or more subsites ofthe ubiquinone binding site can be determined by computationallyevaluating interactions between functional groups, or moieties, of thetest compound and one or more amino acid side chains and/or backboneatoms in the ubiquinone binding site. Typically, a compound which iscapable of participating in stabilizing interactions with a preselectednumber of subsites, preferably without simultaneously participating insignificant destabilizing interactions, is identified as a potentialinhibitor of DHODH. Such a compound will interact with one or moresubsites, preferably with two or more subsites and, more preferably,with three or more subsites.

The invention further provides methods of designing a compound which isa potential inhibitor of DHODH.

The first method includes the steps of (1) identifying one or morefunctional groups capable of interacting with one or more subsites ofthe DHODH ubiquinone binding site; and (2) identifying a scaffold whichpresents the functional group or functional groups identified in step 1in a suitable orientation for interacting with one or more subsites ofthe DHODH ubiquinone binding site. The compound which results fromattachment of the identified functional groups or moieties to theidentified scaffold is a potential inhibitor of DHODH. The DHODHubiquinone binding site is, generally, defined by the atomic coordinatesof a polypeptide comprising the DHODH ubiquinone binding site, forexample, the atomic coordinates set forth in Tables 29, 30, and 31.

The second method comprises the steps of (1) identifying one or morefunctional groups or moieties capable of interacting in a similar way asone or more functional groups or moieties of the co-crystallized ligand,and (2) identifying a scaffold which presents the functional group orfunctional groups identified in step 1 in a suitable orientation forinteracting in a similar way as one or more functional groups ormoieties of the co-crystallized ligand. The compound which results fromattachment of the identified functional groups or moieties to theidentified scaffold is a potential inhibitor of DHODH. Theco-crystallized ligand is, generally, defined by the atomic coordinatesof a ligand complexed in the polypeptide comprising the DHODH ubiquinonebinding site, for example, the atomic coordinates set forth in Tables29, 30, and 31.

Suitable methods, as known in the art, can be used to identify chemicalmoieties, fragments or functional groups which are capable ofinteracting favorably with a particular subsite or sets of subsites.These methods include, but are not limited to: interactive moleculargraphics; molecular mechanics; conformational analysis; energyevaluation; docking; database searching; virtual high-throughputscreening (U.S. Pat. No. 422,303, DE 10009479, EP 1094415, U.S. Pat. No.693,731, U.S. Pat. No. 885,893, U.S. Pat. No. 885,517); structuralalignment; functional group alignment; interaction-point alignment;pharmacophore modeling; de novo design; property estimation anddescriptor-based database searching. These methods can also be employedto assemble chemical moieties, fragments or functional groups into asingle inhibitor molecule. These same methods can also be used todetermine whether a given chemical moiety, fragment or functional groupis able to interact favorably with a particular subsite or sets ofsubsites.

In one embodiment, the design of potential DHODH inhibitors begins fromthe general perspective of three-dimensional shape and electrostaticcomplementarity for the ubiquinone binding site, and subsequently,interactive molecular modeling techniques can be applied by one skilledin the art to visually inspect the quality of the fit of a candidatemolecule into the binding site. Suitable visualization programs includeSYBYL (Tripos Inc., St. Louis, Mo.), MOLOC (Gerber Molecular Design,Basel), RASMOL (Sayle et al. Trends Biochem. Sci. 20:374-376 (1995)) andMOE (Chemical Computing Group Inc., Montreal).

A further embodiment of the present invention utilizes a databasesearching program which is capable of scanning a database of smallmolecules of known three-dimensional structure for candidates which fitinto the target protein site. Suitable software programs include 4SCan®(U.S. Pat. No. 422,303, DE 10009479, EP 1094415, U.S. Pat. No. 693,731,U.S. Pat. No. 885,893, U.S. Pat. No. 885,517), FLEXX (Rarey et al., J.Mol. Biol. 261:470-489 (1996)), and UNITY (Tripos Inc., St. Louis, Mo.).Especially 4SCan® was developed to scan/screen large virtual databasesup to several millions of small molecules in a reasonable time-frame.

A further embodiment of the present invention utilizes a databasesearching program which is capable of scanning a database of smallmolecules of known three-dimensional structure for candidates whichalign properly with the co-crystallized ligand, both in shape andinteraction properties. Suitable software programs include 4SCan® (U.S.Pat. No. 422,303, DE 10009479, EP 1094415, U.S. Pat. No. 693,731, U.S.Pat. No. 885,893, U.S. Pat. No. 885,517) and FLEXS (Lemmen et al., J.Med. Chem 41:4502-4520 (1998)). Especially 4SCan® is capable of aligninglarge virtual databases up to several millions of small molecules in areasonable time-frame.

It is not expected that the molecules found in the search willnecessarily be leads themselves, since a complete evaluation of allinteractions will necessarily be made during the initial search. Rather,it is anticipated that such candidates might act as the framework forfurther design, providing molecular skeletons to which appropriateatomic replacements can be made. Of course, the chemical complementarityof these molecules can be evaluated, but it is expected that thescaffold, functional groups, linkers and/or monomers may be changed tomaximize the electrostatic, hydrogen bonding, and hydrophobicinteractions with the enzyme.

Goodford (Goodford J. Med. Chem. 28:849-857 (1985)) has produced acomputer program, GRID, which seeks to determine regions of highaffinity for different chemical groups (termed probes) on the molecularsurface of the binding site. GRID hence provides a tool for suggestingmodifications to known ligands that might enhance binding.

Consequently, virtual combinatorial libraries covering numerousvariations of the addressed scaffold, functional groups, linkers and/ormonomers can be build up using suitable software programs includingLEGION (Tripos Inc., St. Louis, Mo.) or ACCORD FOR EXCEL (Accelrys Inc.,San Diego, Calif.), followed by scanning or virtual screening or dockingof these libraries using suitable software mentioned above.

A range of factors, including electrostatic interactions, hydrogenbonding, hydrophobic interactions, desolvation effects, conformationalstrain, ligand flexibility and cooperative motions of ligand and enzyme,all influence the binding effect and should be taken into account inattempts to design bioactive inhibitors.

Yet another embodiment of a computer-assisted molecular design methodfor identifying inhibitors of DHODH comprises searching for fragmentswhich fit into a binding region subsite and link to a pre-definedscaffold. The scaffold itself may be identified in such a manner. Arepresentative program suitable for the searching of such functionalgroups and monomers include LUDI (Boehm, J. Comp. Aid. Mol. Des. 6:61-78(1992)) and MCSS (Miranker et al., Proteins 11: 314-328 (1991)).

Yet another embodiment of a computer-assisted molecular design methodfor identifying inhibitors of DHODH comprises the de novo synthesis ofpotential inhibitors by algorithmic connection of small molecularfragments that will exhibit the desired structural and electrostaticcomplementarity with the active site of the enzyme. The methodologyemploys a large template set of small molecules which are iterativelypierced together in a model of the DHODH ubiquinone binding site.Programs suitable for this task include GROW (Moon et al. Proteins11:314-328 (1991)) and SPROUT (Gillet et al. J. Comp. Aid. Mol. Des.7:127 (1993)).

In yet another embodiment, the suitability of inhibitor candidates canbe determined using an empirical scoring function, which can rank thebinding affinities for a set of inhibitors. For examples of such amethod see Muegge et al. and references therein (Muegge et al., J. Med.Chem. 42:791-804 (1999)) and ScoreDock (Tao et al. J. Comp. Aid. Mol.Des. 15: 429-446 (2001)).

Other modeling techniques can be used in accordance with this invention,for example, those described by Stahl (Stahl, in: Virtual Screening forBioactive Molecules, Wiley-VCH, Weinheim, 2000, pp. 229-264), Cohen etal. (J. Med. Chem. 33:883-894 (1990)); Navia et al. (Current Opinions inStructural Biology 2 :202-210 (1992)); Baldwin et al. (J. Med. Chem.32:2510-2513 (1989)); Appelt et al. (J. Med. Chem. 34:1925-1934 (1991));Ealick et al. (Proc. Nat. Acad. Sci. USA 88:11540-11544 (1991));

A compound which is identified by one of the foregoing methods as apotential inhibitor of DHODH can then be obtained, for example, bysynthesis or from a compound library, and assessed for the ability toinhibit DHODH in vitro. Such an in vitro assay can be performed as isknown in the art, for example, by contacting DHODH in solution with thetest compound in the presence of the substrate and cofactor of DHODH andubiquinone. The rate of substrate transformation can be determined inthe presence of the test compound and compared with the rate in theabsence of the test compound. Suitable assays for DHODH biologicalactivity are described below, the teachings of each of which are herebyincorporated by reference herein in their entity.

An inhibitor identified or designed by a method of the present inventioncan be a competitive inhibitor, an uncompetitive inhibitor or anoncompetitive inhibitor with respect to ubiquinone.

A screen of thousands of compounds using 4Scan® as described above wasperformed. Hits were ranked according to consensus score.

In table 25 the structures of the highest ranking compounds of thecombinatorial library are shown. The consensus score of each molecule iscalculated by the summation of the two predicted 4SCan® activity scoresfor the two different structures of the ubiquinone binding site.

The compounds of the present invention can be used for a variety ofhuman and animal diseases, preferably human diseases, where inhibitionof the pyrimidine metabolism is beneficial. Such diseases are:

-   -   fibrosis, uveitis, rhinitis, asthma or arthropathy, in        particular, arthrosis    -   all forms of rheumatism    -   acute immunological events and disorders such as sepsis, septic        shock, endotoxic shock, Gram-negative sepsis, toxic shock        syndrome, acute respiratory distress syndrome, stroke,        reperfusion injury, CNS injury, serious forms of allergy, graft        versus host and host versus graft reactions, alzheimer's disease        or pyresis, restenosis, chronic pulmonary inflammatory disease,        silicosis, pulmonary sarcosis, bone resorption disease. These        immunological events also include a desired modulation and        suppression of the immune system;    -   all types of autoimmune diseases, in particular rheumatoid        arthritis, rheumatoid spondylitis, osteoarthritis, gouty        arthritis, multiple sclerosis, insulin dependent diabetes        mellitus and non-insulin dependent diabetes mellitus, and lupus        erythematoidis, ulcerative colitis, Morbus Crohn, inflammatory        bowel disease, as well as other chronic inflammations, chronic        diarrhea;    -   dermatological disorders such as psoriasis    -   progressive retinal atrophy    -   all kinds of infections including opportunistic infections.

The compounds according to the invention and medicaments preparedtherewith are generally useful for the treatment of cell proliferationdisorders, for the treatment or prophylaxis, immunological diseases andconditions (as for instance inflammatory diseases, neuroimmunologicaldiseases, autoimmune diseases or other).

The compounds of the present invention are also useful for thedevelopment of immunomodulatory and anti-inflammatory medicaments or,more generally, for the treatment of diseases where the inhibition ofthe pyrimidine biosynthesis is beneficial.

The compounds of the present invention are also useful for the treatmentof diseases which are caused by malignant cell proliferation, such asall forms of hematological and solid cancer. Therefore the compoundsaccording to the invention and medicaments prepared therewith aregenerally useful for regulating cell activation, cell proliferation,cell survival, cell differentiation, cell cycle, cell maturation andcell death or to induce systemic changes in metabolism such as changesin sugar, lipid or protein metabolism. They can also be used to supportcell generation poiesis, including blood cell growth and generation(prohematopoietic effect) after depletion or destruction of cells, ascaused by, for example, toxic agents, radiation, immunotherapy, growthdefects, malnutrition, malabsorption, immune dysregulation, anemia andthe like or to provide a therapeutic control of tissue generation anddegradation, and therapeutic modification of cell and tissue maintenanceand blood cell homeostasis.

These diseases and conditions include but are not limited to cancer ashematological (e.g. leukemia, lymphoma, myeloma) or solid tumors (forexample breast, prostate, liver, bladder, lung, esophageal, stomach,colorectal, genitourinary, gastrointestinal, skin, pancreatic, brain,uterine, colon, head and neck, ovarian, melanoma, astrocytoma, smallcell lung cancer, glioma, basal and squameous cell carcinoma, sarcomasas Kaposi's sarcoma and osteosarcoma), treatment of disorders involvingT-cells such as aplastic anemia and DiGeorge syndrome, Graves' disease.

Leflunomide was previously found to inhibit HCMV replication in cellculture. Ocular herpes is the most common cause of infectious blindnessin the developed world. There are about 50.000 cases per year in the USalone, of which 90% are recurrences of initial infections. Recurrencesare treated with antivirals and corticosteroids. Cytomegalovirus,another herpes virus, is a common cause of retinal damage and blindnessin patients with aids. The compounds of the present invention can beused alone or in combination with other antiviral compounds such asganciclovir and foscarnet to treat such diseases.

The compounds of the present invention can further be used for diseasesthat are caused by protozoal infestations in humans and animals. Suchveterinary and human pathogenic protozoas are preferably intracellularactive parasites of the phylum Apicomplexa or Sarcomastigophora,especially Trypanosoma, Plasmodia, Leishmania, Babesia and Theileria,Cryptosporidia, Sacrocystida, Amoebia, Coccidia and Trichomonadia. Theseactive substances or corresponding drugs are especially suitable for thetreatment of Malaria tropica, caused by Plasmodium falciparum, Malariatertiana, caused by Plasmodium vivax or Plasmodium ovale and for thetreatment of Malaria quartana, caused by Plasmodium malariae. They arealso suitable for the treatment of Toxoplasmosis, caused by Toxoplasmagondii, Coccidiosis, caused for instance by Isospora belli, intestinalSarcosporidiosis, caused by Sarcocystis suihominis, dysentery caused byEntamoeba histolytica, Cryptosporidiosis, caused by Cryptosporidiumparvum, Chargas disease, caused by Trypanosoma cruzi, sleeping sickness,caused by Trypanosoma brucei rhodesiense or gambiense, the cutaneous andvisceral as well as other forms of Leishmaniosis. They are also suitablefor the treatment of animals infected by veterinary pathogenic protozoa,like Theileria parva, the pathogen causing bovine East coast fever,Trypanosoma congolense congolense or Trypanosoma vivax vivax,Trypanosoma brucei brucei, pathogens causing Nagana cattle disease inAfrica, Trypanosoma brucei evansi causing Surra, Babesia bigemina, thepathogen causing Texas fever in cattle and buffalos, Babesia bovis, thepathogen causing european bovine Babesiosis as well as Babesiosis indogs, cats and sheep, Sarcocystis ovicanis and ovifelis pathogenscausing Sarcocystiosis in sheep, cattle and pigs, Cryptosporidia,pathogens causing Cryptosporidioses in cattle and birds, Eimeria andIsospora species, pathogens causing Coccidiosis in rabbits, cattle,sheep, goats, pigs and birds, especially in chickens and turkeys. Theuse of the compounds of the present invention is preferred in particularfor the treatment of Coccidiosis or Malaria infections, or for thepreparation of a drug or feed stuff for the treatment of these diseases.This treatment can be prophylactic or curative. In the treatment ofmalaria, the compounds of the present invention may be combined withother anti-malaria agents.

The compounds of the present invention can further be used for viralinfections or other infections caused for instance by Pneumocystiscarinii.

EXAMPLES

1. X-Ray Structure Determination

Expression and Purification

The cDNA encoding for an N-terminally truncated humanDHODH(Met30-Arg396) was amplified by the polymerase chain reaction (PCR)from a human liver cDNA bank (Invitrogen, Groningen). The followingprimers were used to amplify the DHODH gene form the cDNA bank: DHODH-V:5′-GGA ATT CCA TAT GGC CAC GGG AGA (SEQ ID NO:1) TGA GCG-3′ DHODH-R:5′-GCG CGG ATC CTC ACC TCC GAT GAT (SEQ ID NO:2) CTG C-3′

The underlined sequence regions encode for the cutting sites of therestriction enzymes NdeI (DHODH-V) and BamHI (DHODH-R), respectively.The primers are designed such that subcloning using the NdeI and BamHIrestriction sites into a pET-19b vector is possible. The amplified DNAbands were purified and isolated from an agarose gel (QIAquick PCRpurification kit). The band showed the expected length of 1.2 kb. Theisolated PCR fragment was subcloned into a TOPO vector (Invitrogen,Groningen) according to the protocol outlined in the TOPT TA CloningKit. The TOPO vector including the ligated PCR fragment was digestedwith the restriction enzymes NdeI and BamHI (New England Biolabs Inc.)to produce sticky ends. Finally, the fragment was cloned into theNdeI/BamHI sites of a pET-19b vector (Novagen, Madison, Wis.). Thisvector produced the human DHODH(Met30-Arg396) as an N-terminal tenhistidine fusion protein (his10-hDHODH(Met30-Arg396)). The vector wastransformed into chemical competent E. coli BL21(DE3)Gold cells(Stratagene, LaJolla, Calif.). Cells were stored as glycerol stocks at−80° C. until further use.

100 ml LB-medium in 250 ml flasks containing 100 μL freshly preparedampicilline were inoculated with BL21(DE3)Gold cells hosting thepET-19b/hDHODH(Met30-Arg396) construct. Cells were grown overnight at25° C. and constantly vortexed with 150 rpm.

For the expression cultures four 2 L flasks each were filled with 800 mLrich medium (LB) containing 800 μL ampicilline . The flasks wereinoculated with 40 mL of overnight culture and were grown to an opticaldensity O.D.₆₀₀ of 0.6-0.8 at 25° C. The cells were induced with 80 μLof a 1 M isopropyl-β-D-thiogalactoside (IPTG) stock solution and grownfor another 20 h at 25° C.

The cells were harvested by centrifugation for 15 min in a JA-10Beckmann rotor at 5000 rpm at 4° C. The cell pellet was stored untilfurther use at −20° C.

The pellets of 4×800 mL expression were thawed on ice and resuspended in100 mL lysisbuffer containing 50 mM HEPES at pH 7.7, 300 mM NaCl, 10%glycerol, 10% bugbuster (Novagen, 10×), two tablets ofprotease-inhibitor mix (Complete Tabletes EDTA-free, Roche) and 1%triton X-100. The cell suspension was incubated under gentle rocking for20 min at room temperature.

Cell lysis was performed via ultra sonification using a Bransonsonotrode. The chosen parameters for sonification were the following:amplitude: 60% duration: 3 × 3 min maximal allowed temperature: 37° C.pulse duration: 0.5 sec duty cycle: 0.1 sec

The resulting suspension was centrifuged in a JA-25.50 rotor (Beckmann)at 25.000 rpm for 1 hour at 4° C.

The supernatant was loaded onto a Ni-NTA-column (resin was from Quiagen,column adapter from Pharmacia). The column had a bed volume of 3 mL andwas equilibrated with 5 column volumes (CV) of starting buffer (50 mMHEPES pH 7.7; 300 mM NaCl; 10% glycerol and 10 mM imidazole). The samplewas loaded with a flow rate of 1 mL/min at 4° C. using a BioRadEconopump. Then the column was mounted on a BioRad BioLogic-LPchromatography system and washed with 5-10 CVs of 50 mM HEPES pH 7.7,300 mM NaCl, 10% glycerol, 10 mM imidazole and 10 mMN,N-dimethylundecylamin-N-oxide (C11DAO) at a rate of 1 mL/min. Anothermore stringent washing step was performed by applying step gradientsconsisting of the above washing buffer containing 20 mM and 50 mMimidazole, respectively. At this point, pure DHODH was eluted with 50 mMHEPES pH 7.7, 300 mM NaCl, 10% glycerol, 200 mM imidazole and 10 mMN,N-dimethylundecylamin-N-oxide. Elution was carried out with a flowrate of 0.5 mL/min and the eluate was collected in 4 mL fractions.Fractions containing hDHODH(Met30-Arg396) are characterized by a brightyellow colour and showed full activity in an in vitro assay (asdescribed above/below).

Fractions containing hDHODH were combined (approx. 10 mL) and dialysedagainst 3 L of buffer containing 50 mM HEPES pH 7.7, 400 mM NaCl, 30%glycerol, 1 mM EDTA and 10 mM N,N-dimethylundecylamin-N-oxide overnightat 4° C. The dialysed protein sample was concentrated to a finalconcentration of 20 mg/mL using an Ultrafree 4/YM-30 device fromMillipore. During the concentrating procedure the temperature was keptat 4° C. The protein concentration was determined spectrometrically. TheHis-tag was not removed for further studies.

Finally, aliquots of 50 μL were flash frozen in liquid nitrogen andstored at −80° C. until further use.

Crystallization and Data Collection

Human his10-hDHODH(Met30-Arg396) was co-crystallized with compound 1 andcompound 2 at 20° C. using the hanging-drop vapour diffusion method.Drops were formed by mixing equal amounts of 20 mg/ml protein in 50 mMHEPES pH 7.7, 400 mM NaCl, 30% glycerol, 1 mM EDTA and 10 mMN,N-dimethylundecylamin-N-oxide (C11DAO) with a precipitant solution of0.1 M acetate pH 4.6-5.0, 40 mM C11DAO, 20.8 mMN,N-dimethyldecylamine-N-oxide (DDAO), 2 mM dihydroorotate (DHO),1.8-2.4 M ammonium sulfate, 1 mM compound 1 or 2. The hanging drops wereincubated against 0.5 mL reservoir of 0.1 M acetate pH 4.8, 2.4-2.6 Mammonium sulfate and 30% glycerol. The crystallization conditions werescreened by variation of pH versus ammonium sulfate concentration usinga small grid screen (see FIG. 2):

The same procedure was applied to obtain single crystals ofDHODH(Met30-Arg396) in complex with compounds 3, 4, 5, 6, 7, 8, 9 and10. Compounds 5 and 6 were synthesized as racemic mixtures due to thepresence of a stereo center at the five membered ring. The racemicmixtures were used for crystallization experiments.

Crystals usually appeared as small cubes within three days. They usuallyreached a full size of 0.2×0.2×0.2 mm within three to four weeks. Theprotein crystallized in the space group P3₂21. Crystals were harvestedwith pre-mounted loops of size 0.5 mm (Hampton Research) and were flashfrozen directly in the cryo stream of the measurement device.

Data were collected at the beamline BW6 at the DESY Hamburg on a MAR-CCDcamera. A total of 120 frames (0.5° each) were collected from a humanDHODH(Met30-Arg396) crystal co-crystallized with compound 1. For thecrystal cocrystallized with compound 2 a total of 85 frames (1° each)was recorded. The crystals were maintained at a temperature of 100 Kduring data collection. The indexing and integration of the reflectionintensities were performed with the program MOSFLM (CollaborativeComputational Project, Number 4 (1994). Acta Cryst. D50, 760-763.). Datawere scaled and merged with SCALA and reduced to structure factoramplitudes with TRUNCATE, both from the CCP4 program suite(Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50,760-763.). At this stage 5% and 10% (the “test set”) of uniquereflections were flagged for cross validation to calculate the freeR-factor (R_(free)) during the refinement process later on for compound1 and compound 2, respectively. The remaining 95% and 90% of thereflections constituted the “working set” for calculation of theR-factor (R), respectively. The statistics of data collection are shownin table 1 and table 2. TABLE 1 Crystal & Data collection statistics forcompound 1 A. Crystal data Spacegroup P3₂21 Cell dimensions (Å) a =90.69 b = 90.69 c = 123.22 Molecules/asymmetric unit 1 Matthews'constant (V_(m))(Å³/Da) 4.1 Maximum resolution (Å) 2.35 B. DataCollection X-Ray source DESY BW6 Wavelength (Å) 1.05 Total/uniquereflections 91431/24977 Completeness (%) 98.2 (99.0) I/sigma 23.9 (6.5)R_(merge) (%) 5.7 (20.2)

TABLE 2 Crystal & Data collection statistics for compound 2 A. Crystaldata Spacegroup P3₂21 Cell dimensions (Å) a = 90.65 b = 90.65 c = 123.07Molecules/asymmetric unit 1 Matthews' constant (V_(m))(Å³/Da) 4.1Maximum resolution (Å) 2.4 B. Data Collection X-Ray source DESY BW6Wavelength (Å) 1.05 Total/unique reflections 101935/22253 Completeness(%) 95.8 (97.1) I/sigma 14.6 (3.8) R_(merge) (%) 9.1 (38.1)

Datasets for the crystals of human DHODH(Met30-Arg396) co-crystallizedwith compounds 3, 4, 6, 7, 8, 9 and 10 were also collected at thebeamline BW6 at the DESY Hamburg on a MAR-CCD camera. Co-crystals withcompound 5 were recorded at an in house generator using CuKα radiationand a MAR-dtb image plate.

A total of 55 frames, 65 frames, 96 frames, 62 frames, 120 frames, 60frames, 100 frames and 100 frames (1° each) were collected from humanDHODH(Met30-Arg396) crystals co-crystallized with compound 3, 4, 5, 6,7, 8, 9 and 10 respectively. The crystals were maintained at atemperature of 100 K during data collection. The indexing andintegration of the reflection intensities were performed with theprogram MOSFLM (Collaborative Computational Project, Number 4 (1994).Acta Cryst. D50, 760-763.). Data were scaled and merged with SCALA andreduced to structure factor amplitudes with TRUNCATE, both from the CCP4program suite (Collaborative Computational Project, Number 4 (1994).Acta Cryst. D50, 760-763.). At this stage 5% or 10% (the “test set”) ofunique reflections were flagged for cross validation to calculate thefree R-factor (R_(free)) during the refinement process. The remaining95% or 90% of the reflections constituted the “working set” forcalculation of the R-factor (R), respectively. The statistics of datacollection are shown in tables 5 to 12, respectively. TABLE 5 Crystal &Data collection statistics for compound 3 A. Crystal data SpacegroupP3₂21 Cell dimensions (Å) a = 90.43 b = 90.43 c = 123.00Molecules/asymmetric unit 1 Matthews' constant (V_(m))(Å³/Da) 4.1Maximum resolution (Å) 1.95 B. Data Collection X-Ray source DESY BW6Wavelength (Å) 1.05 Total/unique reflections 142628/42908 Completeness(%) 99.8/99.9 I/sigma 12.6/3.4 R_(merge) (%) 8.2/38.3

TABLE 6 Crystal & Data collection statistics for compound 4 A. Crystaldata Spacegroup P3₂21 Cell dimensions (Å) a = 90.65 b = 90.65 c = 123.21Molecules/asymmetric unit 1 Matthews' constant (V_(m))(Å³/Da) 4.1Maximum resolution (Å) 2.15 B. Data Collection X-Ray source DESY BW6Wavelength (Å) 1.05 Total/unique reflections 124056/32175 Completeness(%) 99.2/99.0 I/sigma 14.7/5.7 R_(merge) (%) 7.1/24.8

TABLE 7 Crystal & Data collection statistics for compound 5 A. Crystaldata Spacegroup P3₂21 Cell dimensions (Å) a = 90.30 b = 90.30 c = 123.09Molecules/asymmetric unit 1 Matthews' constant (V_(m))(Å³/Da) 4.1Maximum resolution (Å) 2.2 B. Data Collection X-Ray source CuKαWavelength (Å) 1.54 Total/unique reflections 171127/30057 Completeness(%) 99.9 (99.9) I/sigma 4.0/1.9 R_(merge) (%) 15.4/43.5

TABLE 8 Crystal & Data collection statistics for compound 6 A. Crystaldata Spacegroup P3₂21 Cell dimensions (Å) a = 90.44 b = 90.44 c = 123.20Molecules/asymmetric unit 1 Matthews' constant (V_(m))(Å³/Da) 4.1Maximum resolution (Å) 1.9 B. Data Collection X-Ray source DESY BW 6Wavelength (Å) 1.05 Total/unique reflections 173775/46257 Completeness(%) 99.4/99.9 I/sigma 13.8/2.8 R_(merge) (%) 8.5/46.0

TABLE 9 Crystal & Data collection statistics for compound 7 A. Crystaldata Spacegroup P3₂21 Cell dimensions (Å) a = 90.74 b = 90.74 c = 122.88Molecules/asymmetric unit 1 Matthews' constant (V_(m))(Å³/Da) 4.1Maximum resolution (Å) 1.9 B. Data Collection X-Ray source DESY BW 6Wavelength (Å) 1.05 Total/unique reflections 341319/46198 Completeness(%) 98.6/99.7 I/sigma 23.5/5.1 R_(merge) (%) 8.2/21.8

TABLE 10 Crystal & Data collection statistics for compound 8 A. Crystaldata Spacegroup P3₂21 Cell dimensions (Å) a = 90.56 b = 90.56 c = 123.06Molecules/asymmetric unit 1 Matthews' constant (V_(m))(Å³/Da) 4.1Maximum resolution (Å) 1.8 B. Data Collection X-Ray source DESY BW 6Wavelength (Å) 1.05 Total/unique reflections 190208/53993 Completeness(%) 98.8/96.7 I/sigma 16.7/2.9 R_(merge) (%) 6.3/38.3

TABLE 11 Crystal & Data collection statistics for compound 9 A. Crystaldata Spacegroup P3₂21 Cell dimensions (Å) a = 90.29 b = 90.29 c = 122.69Molecules/asymmetric unit 1 Matthews' constant (V_(m))(Å³/Da) 4.1Maximum resolution (Å) 2.0 B. Data Collection X-Ray source DESY BW 6Wavelength (Å) 1.05 Total/unique reflections 103711/39080 Completeness(%) 98.6/99.0 I/sigma 14.1/3.9 R_(merge) (%) 6.5/24.8

TABLE 12 Crystal & Data collection statistics for compound 10 A. Crystaldata Spacegroup P3₂21 Cell dimensions (Å) a = 90.75 b = 90.75 c = 122.71Molecules/asymmetric unit 1 Matthews' constant (V_(m))(Å³/Da) 4.1Maximum resolution (Å) 1.8 B. Data Collection X-Ray source DESY BW 6Wavelength (Å) 1.05 Total/unique reflections 326425/54728 Completeness(%) 99.9/100 I/sigma 27.5/6.0 R_(merge) (%) 6.0/30.6Structure Determination and Refinement of DHODH/Compound 1 Complex

The structure for the human DHODH(Met30-Arg396) in complex with compound1 was solved using the method of molecular replacement (MR). The freeaccessible pdb entry 1D3G.pdb was used as a search model. The ligandsbrequinar and DDQ as well as all of the water molecules were removedprior to the MR search. The search model included the polypeptide chainof hDHODH(Met30-Arg396), one molecule of orotate, one molecule of thecofactor flavinmononucleotide (FMN) and one acetate molecule which waspresent under the crystallization conditions. A standard rotational andtranslational molecular replacement search at 3.5 Å was performed usingthe program molrep (Collaborative Computational Project, Number 4(1994). Acta Cryst. D50, 760-763.). Solutions for both the rotationaland translational search were well above the next ranking solutions. TheMR resulted in an R-factor of 35.6% and a correlation coefficient of69.4% for compound 1 complex.

In a first round of refinement the MR model was subjected to rigid bodyrefinement and a slow cooling simulated annealing protocol using amaximum likelihood target to remove model bias (Accelrys Inc. CNXprogram suite, CNX2002). Additionally, an individual b-factor refinementwas carried out using standard CNX protocols. Finally, SIGMAA weighted2Fo-Fc and Fo-Fc electron density maps were calculated and displayedtogether with the protein model in the program 0 (DatOno A B; Jones, T.A., Zou, J. Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,110-119.). The resulting experimental electron density was so excellentthat the conformation of compound 1 could be interpreted unambiguously.

A pdb file for compound 1 was created using the program MOE (ChemicalComputing Group Inc., MOE 2002.02). After energy minimization thecompound was built into the electron density manually. Topology andparameter files for compound 1 were created using the program Xplo2d(Uppsala Software Factory; Kleywegt, G. M. (1997) J. Mol. Biol. 273,371-376). After an additional round of model building and water pickingusing CNX another complete round of refinement was performed. The finalmodel included the DHODH(Met30-Arg396) protein, the cofactorflavinmononucleotide (FMN), one orotate molecule (ORO), one acetatemolecule (ACT), two sulfate ions (SO₄), one molecule of compound 1 (INH)and 153 water molecules (TIP) (see Table 29). The model is well refinedand has very good geometry. The refinement process which included datafrom 12.0-2.35 Å resulted in an R-factor of 18.5% and a free R-factor of21.7%. With the exception of glycine residues, 92.4% (278) of theresidues are located in the most favoured region of the ramachandranplot and 7.6% (22) cluster in the additional allowed regions. Table 13summarizes the refinement statistics for the inhibitor compound 1 incomplex with human DHODH. Values in parentheses give the R-factor andR_(free)-factors, respectively, for the last resolution bin ranging from2.50 to 2.35.

The N-terminal His tag could not be detected in the electron densitymap. TABLE 13 Refinement Statistics for DHODH/compound 1 complexR-factor (%) 18.5 (19.6) R_(free) 21.7 (24.2) RMS deviation from idealvalues bond length (Å) 0.006 Bond angle (°) 1.2 Dihedral angles (°) 21.4Improper angles (°) 0.83Structure Determination and Refinement of DHODH/Compound 2 Complex

The structure for the human DHODH(Met30-Arg396) in complex with compound2 was solved using the method of molecular replacement (MR). The freeaccessible pdb entry 1D3G.pdb was used as a search model. The ligandsbrequinar and DDQ as well as all of the water molecules were removedprior to the MR search. The search model included the polypeptide chainof hDHODH(Met30-Arg396), one molecule of orotate, one molecule of thecofactor flavinmononucleotide (FMN) and one acetate molecule which waspresent under the crystallization conditions. A standard rotational andtranslational molecular replacement search at 3.5 Å was performed usingthe program molrep (Collaborative Computational Project, Number 4(1994). Acta Cryst. D50, 760-763.). Solutions for both the rotationaland translational search were well above the next ranking solutions. TheMR resulted in an R-factor of 33.8% and a correlation coefficient of68.2% for the DHODH/compound 2 complex.

In a first round of refinement the MR model was subjected to rigid bodyrefinement and a slow cooling simulated annealing protocol using amaximum likelihood target to remove model bias (Accelrys Inc. CNXprogram suite, CNX2002). Additionally, an individual b-factor refinementwas carried out using standard CNX-protocols. Finally a SIGMAA weighted2Fo-Fc and Fo-Fc electron density maps were calculated and displayedtogether with the protein model in the program O (DatOno A B; Jones, T.A., Zou, J. Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,110-119.). The resulting experimental electron density was so excellentthat the conformation of the inhibitor compound 2 could be interpretedunambiguously. The electron density around the five-membered ringcarrying the carboxy group clearly showed the presence of twoalternative conformations of compound 2. In one conformation(conformation A) the carboxy group interacts with residues Gln 47 andArg 136, whereas in the second conformation (conformation B) theinteraction involves residues His 56 and Tyr 356 (see above). For eachconformation a separate DHODH/compound 2 complex was subjected torefinement.

Pdb files for the compound 2 in conformation A and B were created usingthe program MOE (Chemical Computing Group Inc., MOE 2002.02). Bothcompounds were energy minimized and built into the electron densitymanually. Topology and parameter files for compound 2 were created usingthe program Xplo2d (Uppsala Software Factory; Kleywegt, G. M. (1997) J.Mol. Biol., 273, 371-376). After an additional round of model buildingand water picking using CNX, another complete round of refinement wasperformed. The final model included the human DHODH(Met30-Arg396)protein, the cofactor flavinmononucleotide (FMN), one orotate molecule(ORO), one acetate molecule (ACT), four sulfate ions (SO4), one moleculeof compound 2 (INH) either in conformation A or conformation B and 250water molecules (TIP) (see Tables 30 and 31). The models are wellrefined and show very good geometry. The refinement process whichincluded data from 12.0-2.4 Å resulted in an R-factor of 17.5% and afree R-factor of 21.1% for conformation A complex and an R-factor of17.6% and a free R-factor of 21.6% for conformation B complex,respectively. With the exception of glycine residues, 91.7% (276) of theresidues are located in the most favoured region of the ramachandranplot and 8.3% (24) cluster in the additional allowed regions. Table 14summarizes the refinement statistics for compound 2 in complex withhuman DHODH. Values in parentheses give the R-factor andR_(free)-factors, respectively, for the last resolution bin ranging from2.55 to 2.40. TABLE 14 Refinement Statistics for DHODH/compound 2complex Conformation A Conformation B R-factor (%) 17.5 (19.6) 17.6(19.4) R_(free) 21.1 (23.6) 21.6 (23.2) RMS deviation from ideal valuesbond length (Å) 0.005 0.005 Bond angle (°) 1.2 1.2 Dihedral angles (°)21.3 21.3 Improper angles (°) 0.81 0.81Structure Determination and Refinement of DHODH/Compound 3 Complex

The structure for the human DHODH(Met30-Arg396) in complex with compound3 was solved using the method of molecular replacement (MR). The freeaccessible pdb entry 1D3G.pdb was used as a search model. The ligandsbrequinar and DDQ as well as all of the water molecules were removedprior to the MR search. The search model included the polypeptide chainof hDHODH(Met30-Arg396), one molecule of orotate, one molecule of thecofactor flavinmononucleotide (FMN) and one acetate molecule which waspresent under the crystallization conditions. A standard rotational andtranslational molecular replacement search at 3.0 Å was performed usingthe program molrep (Collaborative Computational Project, Number 4(1994). Acta Cryst. D50, 760-763.). Solutions for both the rotationaland translational search were well above the next ranking solutions. TheMR resulted in an R-factor of 33.9% and a correlation coefficient of72.5 for the DHODH/compound 3 complex.

In a first round of refinement the MR model was subjected to rigid bodyrefinement and a slow cooling simulated annealing protocol using amaximum likelihood target to remove model bias (Accelrys Inc. CNXprogram suite, CNX2002). Additionally, an individual b-factor refinementwas carried out using standard CNX-protocols. Finally SIGMAA weighted2Fo-Fc and Fo-Fc electron density maps were calculated and displayedtogether with the protein model in the program O (DatOno A B; Jones, T.A., Zou, J. Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,110-119.). The resulting experimental electron density was so excellentthat the conformation of the inhibitor compound 3 could be interpretedunambiguously. The electron density around the five-membered ringcarrying the carboxy group clearly showed the presence of twoalternative conformations of compound 3. In one conformation(conformation A) the carboxy group interacts with residues Gln 47 andArg 136, whereas in the second conformation (conformation B) theinteraction involves residues His 56 and Tyr 356 (see above). For eachconformation a separate DHODH/compound 3 complex was subjected torefinement.

The pdb files for the compound 3 in conformation A and B were createdusing the program MOE (Chemical Computing Group Inc., MOE 2002.02). Bothcompounds were energy minimized and built into the electron densitymanually. Topology and parameter files for compound 3 were created usingthe program Xplo2d (Uppsala Software Factory; Kleywegt, G. M. (1997) J.Mol. Biol., 273, 371-376). After an additional round of model buildingand water picking using CNX, another complete round of refinement wasperformed. The final model included the human DHODH(Met30-Arg396)protein, the cofactor flavinmononucleotide (FMN), one orotate molecule(ORO), two acetate molecules (ACT), two sulfate ions (SO4), one moleculeof compound 3 (INH) either in conformation A or conformation B and 263water molecules (WAT). Residues which are missing the coordinate filedue to very poor electron density are listed in the header of the pdbfiles.

The models are well refined and show very good geometry. The refinementprocess which included data from 19.9-1.95 Å resulted in an R-factor of18.5% and a free R-factor of 20.3% for the complex in conformation A andan R-factor of 18.5% and a free R-factor of 20.3% for the complex inconformation B, respectively. The almost identical R-factors indicatethat non of the conformers A and B represent a preferred conformation.Except for non-glycine and non-proline residues 91.6% are located in themost favoured region of the ramachandran plot and 8% cluster in theadditional allowed regions. There are no residues in the disallowedregion. Table 15 summarizes the refinement statistics for compound 3 incomplex with human DHODH. Values in parentheses give the R-factor andR_(free)-factors, respectively, for the last resolution bin ranging from2.07 to 1.95. TABLE 15 Refinement Statistics for DHODH/compound 3complex conformation A conformation B R-factor (%) 18.5 (20.6) 18.5(20.6) R_(free) 20.3 (23.5) 20.2 (23.6) RMS deviation from ideal valuesbond length (Å) 0.005 0.005 Bond angle (°) 1.2 1.2 Dihedral angles (°)21.2 21.2 Improper angles (°) 0.81 0.81Structure Determination and Refinement of DHODH/Compound 4 Complex

The structure for the human DHODH(Met30-Arg396) in complex with compound4 was solved using the method of molecular replacement (MR). The freeaccessible pdb entry 1D3G.pdb was used as a search model. The ligandsbrequinar and DDQ as well as all of the water molecules were removedprior to the MR search. The search model included the polypeptide chainof hDHODH(Met30-Arg396), one molecule of orotate, one molecule of thecofactor flavinmononucleotide (FMN) and one acetate molecule which waspresent under the crystallization conditions. A standard rotational andtranslational molecular replacement search at 3.0 Å was performed usingthe program molrep (Collaborative Computational Project, Number 4(1994). Acta Cryst. D50, 760-763.). Solutions for both the rotationaland translational search were well above the next ranking solutions. TheMR resulted in an R-factor of 34.6% and a correlation coefficient of71.1 for the DHODH/compound 4 complex.

In a first round of refinement the MR model was subjected to rigid bodyrefinement and a slow cooling simulated annealing protocol using amaximum likelihood target to remove model bias (Accelrys Inc. CNXprogram suite, CNX2002). Additionally, an individual b-factor refinementwas carried out using standard CNX-protocols. Finally SIGMAA weighted2Fo-Fc and Fo-Fc electron density maps were calculated and displayedtogether with the protein model in the program O (DatOno A B; Jones, T.A., Zou, J. Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,110-119.). The resulting experimental electron density was so excellentthat the conformation of the inhibitor compound 4 could be interpretedunambiguously. The electron density around the five-membered ringcarrying the carboxy group clearly showed the carboxy group in contactwith residues His 56 and Tyr 356 in non-brequinar-like conformation.

A pdb file for compound 4 was created using the program MOE (ChemicalComputing Group Inc., MOE 2002.02). After energy minimization thecompound was built into the electron density manually. Topology andparameter files for compound 4 were created using the program Xplo2d(Uppsala Software Factory; Kleywegt, G. M. (1997) J. Mol. Biol. 273,371-376). After an additional round of model building and water pickingusing CNX another complete round of refinement was performed. The finalmodel included the DHODH(Met30-Arg396) protein, the cofactorflavinmononucleotide (FMN), one orotate molecule (ORO), one acetatemolecule (ACT), one sulfate ion (SO₄), one molecule of compound 4 (INH)and 192 water molecules (TIP).

The model is well refined and shows very good stereochemical geometry.The refinement process which included data from 19.9-2.15 Å resulted inan R-factor of 20.1% and a free R-factor of 22.1%. Except fornon-glycine and non-proline residues 91.6% of the residues are locatedin the most favoured region of the ramachandran plot and 8% and 0.3%cluster in the additional allowed or generously allowed regions,respectively. There are no residues in the disallowed region. Table 16summarizes the refinement statistics for compound 4 in complex withhuman DHODH. Values in parentheses give the R-factor andR_(free)-factors, respectively, for the last resolution bin ranging from2.28 to 2.15. TABLE 16 Refinement Statistics for DHODH/compound 4complex R-factor (%) 20.1 (19.1) R_(free) 22.1 (20.9) RMS deviation fromideal values bond length (Å) 0.005 Bond angle (°) 1.2 Dihedral angles(°) 21.5 Improper angles (°) 0.80Structure Determination and Refinement of DHODH/Compound 5 Complex

The structure for the human DHODH(Met30-Arg396) in complex with compound5 was solved using the method of molecular replacement (MR). The freeaccessible pdb entry 1D3G.pdb was used as a search model. The ligandsbrequinar and DDQ as well as all of the water molecules were removedprior to the MR search. The search model included the polypeptide chainof hDHODH(Met30-Arg396), one molecule of orotate, one molecule of thecofactor flavinmononucleotide (FMN) and one acetate molecule which waspresent under the crystallization conditions. A standard rotational andtranslational molecular replacement search at 3.0 Å was performed usingthe program molrep (Collaborative Computational Project, Number 4(1994). Acta Cryst. D50, 760-763.). Solutions for both the rotationaland translational search were well above the next ranking solutions. TheMR resulted in an R-factor of 33.8% and a correlation coefficient of71.5 for the DHODH/compound 5 complex.

In a first round of refinement the MR model was subjected to rigid bodyrefinement and a slow cooling simulated annealing protocol using amaximum likelihood target to remove model bias (Accelrys Inc. CNXprogram suite, CNX2002). Additionally, an individual b-factor refinementwas carried out using standard CNX-protocols. Finally SIGMAA weighted2Fo-Fc and Fo-Fc electron density maps were calculated and displayedtogether with the protein model in the program O (DatOno A B; Jones, T.A., Zou, J. Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,110-119.). The resulting experimental electron density was so excellentthat the conformation of the inhibitor compound 5 could be interpretedunambiguously. The electron density around the five-membered ringcarrying the carboxy group clearly showed the carboxy group in contactwith residues His 56 and Tyr 356 in non-brequinar-like conformation.Interestingly the protein's active site discriminates between the S- andR-enantiomere. Inspection of the corresponding electron densityunequivocally shows the presences of the R-enantiomere only.

A pdb file for compound 5 was created using the program MOE (ChemicalComputing Group Inc., MOE 2002.02). After energy minimization thecompound was built into the electron density manually. Topology andparameter files for compound 5 were created using the program Xplo2d(Uppsala Software Factory; Kleywegt, G. M. (1997) J. Mol. Biol. 273,371-376). After an additional round of model building and water pickingusing CNX another complete round of refinement was performed. The finalmodel included the DHODH(Met30-Arg396) protein, the cofactorflavinmononucleotide (FMN), one orotate molecule (ORO), one acetatemolecule (ACT), two sulfate ions (SO₄), one molecule of compound 5 (INH)and 287 water molecules (TIP).

The model is well refined and shows very good stereochemical geometry.The refinement process which included data from 25.5-2.2 Å resulted inan R-factor of 18.3% and a free R-factor of 20.9%. Except fornon-glycine and non-proline residues 92.6% of the residues are locatedin the most favoured region of the ramachandran plot and 7% and 0.3%cluster in the additional allowed or generously allowed regions,respectively. There are no residues in the disallowed region. Table 17summarizes the refinement statistics for compound 5 in complex withhuman DHODH. Values in parentheses give the R-factor andR_(free)-factors, respectively, for the last resolution bin ranging from2.34 to 2.2. TABLE 17 Refinement Statistics for DHODH/compound 5 complexR-factor (%) 18.3 (19.4) R_(free) 20.9 (22.0) RMS deviation from idealvalues bond length (Å) 0.005 Bond angle (°) 1.2 Dihedral angles (°) 21.3Improper angles (°) 0.83Structure Determination and Refinement of DHODH/Compound 6 Complex

The structure for the human DHODH(Met30-Arg396) in complex with compound6 was solved using the method of molecular replacement (MR). The freeaccessible pdb entry 1D3G.pdb was used as a search model. The ligandsbrequinar and DDQ as well as all of the water molecules were removedprior to the MR search. The search model included the polypeptide chainof hDHODH(Met30-Arg396), one molecule of orotate, one molecule of thecofactor flavinmononucleotide (FMN) and one acetate molecule which waspresent under the crystallization conditions. A standard rotational andtranslational molecular replacement search at 3.0 Å was performed usingthe program molrep (Collaborative Computational Project, Number 4(1994). Acta Cryst. D50, 760-763.). Solutions for both the rotationaland translational search were well above the next ranking solutions. TheMR resulted in an R-factor of 32.7% and a correlation coefficient of74.5 for the DHODH/compound 6 complex.

In a first round of refinement the MR model was subjected to rigid bodyrefinement and a slow cooling simulated annealing protocol using amaximum likelihood target to remove model bias (Accelrys Inc. CNXprogram suite, CNX2002). Additionally, an individual b-factor refinementwas carried out using standard CNX-protocols. Finally SIGMAA weighted2Fo-Fc and Fo-Fc electron density maps were calculated and displayedtogether with the protein model in the program O (DatOno A B; Jones, T.A., Zou, J. Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,110-119.). The resulting experimental electron density was so excellentthat the conformation of the inhibitor compound 6 could be interpretedunambiguously. The electron density around the five-membered ringcarrying the carboxy group clearly showed that the inhibitor moleculeadopts both the brequinar and non-brequinar binding mode. The carboxygroup is in contact with both anion binding sites. Interestingly theprotein's active site discriminates between the S- and R-enantiomere.Inspection of the corresponding electron density unequivocally shows thepresences of the R-enantiomere only.

A pdb file for compound 6 was created using the program MOE (ChemicalComputing Group Inc., MOE 2002.02). After energy minimization thecompound was built into the electron density manually. Topology andparameter files for compound 6 were created using the program Xplo2d(Uppsala Software Factory; Kleywegt, G. M. (1997) J. Mol. Biol. 273,371-376). After an additional round of model building and water pickingusing CNX another complete round of refinement was performed. The finalmodel included the DHODH(Met30-Arg396) protein, the cofactorflavinmononucleotide (FMN), one orotate molecule (ORO), one acetatemolecule (ACT), one sulfate ion (SO₄), one molecule of compound 6 (INH)and 312 water molecules (TIP).

The models are well refined and show very good geometry. The refinementprocess which included data from 19.3-1.9 Å resulted in an R-factor of18.5% and a free R-factor of 20.8% for the complex in conformation A andan R-factor of 18.5% and a free R-factor of 20.7% for the complex inconformation B, respectively. The almost identical R-factors indicatethat non of the conformers A and B represent a preferred conformation.Except for non-glycine and non-proline residues 92.6% are located in themost favoured region of the ramachandran plot and 7.4% cluster in theadditional allowed regions. There are no residues in the disallowedregion. Table 18 summarizes the refinement statistics for compound 6 incomplex with human DHODH. Values in parentheses give the R-factor andR_(free)-factors, respectively, for the last resolution bin ranging from2.02 to 1.9. TABLE 18 Refinement Statistics for DHODH/compound 6 complexconformation A conformation B R-factor (%) 18.5 (21.1) 18.5 (21.2)R_(free) 20.8 (21.5) 20.7 (21.6) RMS deviation from ideal values bondlength (Å) 0.005 0.005 Bond angle (°) 1.2 1.2 Dihedral angles (°) 21.321.3 Improper angles (°) 0.79 0.79Structure Determination and Refinement of DHODH/Compound 7 Complex

The structure for the human DHODH(Met30-Arg396) in complex with compound7 was solved using the method of molecular replacement (MR). The freeaccessible pdb entry 1D3G.pdb was used as a search model. The ligandsbrequinar and DDQ as well as all of the water molecules were removedprior to the MR search. The search model included the polypeptide chainof hDHODH(Met30-Arg396), one molecule of orotate, one molecule of thecofactor flavinmononucleotide (FMN) and one acetate molecule which waspresent under the crystallization conditions. A standard rotational andtranslational molecular replacement search at 3.0 Å was performed usingthe program molrep (Collaborative Computational Project, Number 4(1994). Acta Cryst. D50, 760-763.). Solutions for both the rotationaland translational search were well above the next ranking solutions. TheMR resulted in an R-factor of 32.7% and a correlation coefficient of73.9 for the DHODH/compound 7 complex.

In a first round of refinement the MR model was subjected to rigid bodyrefinement and a slow cooling simulated annealing protocol using amaximum likelihood target to remove model bias (Accelrys Inc. CNXprogram suite, CNX2002). Additionally, an individual b-factor refinementwas carried out using standard CNX-protocols. Finally SIGMAA weighted2Fo-Fc and Fo-Fc electron density maps were calculated and displayedtogether with the protein model in the program O (DatOno A B; Jones, T.A., Zou, J. Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,110-119.). The resulting experimental electron density was so excellentthat the conformation of the inhibitor compound 7 could be interpretedunambiguously. The electron density around the five-membered ringcarrying the carboxy group clearly showed the carboxy group in contactwith residues His 56 and Tyr 356 in non-brequinar-like conformationaddressing subsite 3. In compound 7 a hydroxy group at 3-position at thefive membered ring was introduced creating a stereo center at thisposition. The racemic mixture was used for crystallization experiments.Analysis of the electron density reveals the presence of bothenantiomeres. Interestingly only the R-enantiomere is able to formadditional contacts to the side chains of residues Gln47 and Arg136 andto a conserved water molecule. As is clearly shown from experimentaldata compound 7 is able to form interactions with both subsite 2 andsubsite 3 at the same time. This feature clearly discriminates thiscompound class from, for example, compounds 2, 6 and 10 which canaddress both binding sites utilizing alternative conformations but notat the same time.

A pdb file for compound 7 was created using the program MOE (ChemicalComputing Group Inc., MOE 2002.02). After energy minimization thecompound was built into the electron density manually. Topology andparameter files for compound 7 were created using the program Xplo2d(Uppsala Software Factory; Kleywegt, G. M. (1997) J. Mol. Biol. 273,371-376). After an additional round of model building and water pickingusing CNX another complete round of refinement was performed. The finalmodel included the DHODH(Met30-Arg396) protein, the cofactorflavinmononucleotide (FMN), one orotate molecule (ORO), one acetatemolecule (ACT), two sulfate ions (SO₄), one molecule of compound 7 (INH)and 229 water molecules (TIP).

The model is well refined and shows very good stereochemical geometry.The refinement process which included data from 17.0-2.0 Å resulted inan R-factor of 17.5% and a free R-factor of 20.4% for the R-form andS-form. Except for non-glycine and non-proline residues 92.3% of theresidues are located in the most favoured region of the ramachandranplot and 7.7% cluster in the additional allowed regions. There are noresidues in the disallowed region. Table 19 summarizes the refinementstatistics for compound 7 in complex with human DHODH. Values inparentheses give the R-factor and R_(free)-factors, respectively, forthe last resolution bin ranging from 2.13 to 2.0. TABLE 19 RefinementStatistics for DHODH/compound 7 complex R-form S-form R-factor (%) 17.5(17.3) 17.5 (17.3) R_(free) 20.4 (21.4) 20.4 (21.4) RMS deviation fromideal values bond length (Å) 0.005 0.008 Bond angle (°) 1.2 1.2 Dihedralangles (°) 21.2 21.2 Improper angles (°) 0.82 0.81Structure Determination and Refinement of DHODH/Compound 8 Complex

The structure for the human DHODH(Met30-Arg396) in complex with compound8 was solved using the method of molecular replacement (MR). The freeaccessible pdb entry 1D3G.pdb was used as a search model. The ligandsbrequinar and DDQ as well as all of the water molecules were removedprior to the MR search. The search model included the polypeptide chainof hDHODH(Met30-Arg396), one molecule of orotate, one molecule of thecofactor flavinmononucleotide (FMN) and one acetate molecule which waspresent under the crystallization conditions. A standard rotational andtranslational molecular replacement search at 3.0 Å was performed usingthe program molrep (Collaborative Computational Project, Number 4(1994). Acta Cryst. D50, 760-763.). Solutions for both the rotationaland translational search were well above the next ranking solutions. TheMR resulted in an R-factor of 33.3% and a correlation coefficient of73.9 for the DHODH/compound 8 complex.

In a first round of refinement the MR model was subjected to rigid bodyrefinement and a slow cooling simulated annealing protocol using amaximum likelihood target to remove model bias (Accelrys Inc. CNXprogram suite, CNX2002). Additionally, an individual b-factor refinementwas carried out using standard CNX-protocols. Finally SIGMAA weighted2Fo-Fc and Fo-Fc electron density maps were calculated and displayedtogether with the protein model in the program O (DatOno A B; Jones, T.A., Zou, J. Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,110-119.). The resulting experimental electron density was so excellentthat the conformation of the inhibitor compound 8 could be interpretedunambiguously. The electron density around the five-membered ringcarrying the carboxy group clearly showed the carboxy group in contactwith residues His 56 and Tyr 356 in non-brequinar-like conformationaddressing subsite 3. In compound 8 a hydroxy group at 5-position at thefive membered ring was introduced creating a stereo center at thisposition. The racemic mixture was used for crystallization experiments.Analysis of the electron density reveals that both enantiomeres fit intothe electron density. The R-enantiomere appears to be positioned in amore favourable position to form interactions with subsite 3 whereas inthe S-enantiomere the hydroxy group protrudes into the direction ofsubsite 4 (remote hydrophobic pocket) in a less favourable manner.

A pdb file for compound 8 was created using the program MOE (ChemicalComputing Group Inc., MOE 2002.02). After energy minimization thecompound was built into the electron density manually. Topology andparameter files for compound 8 were created using the program Xplo2d(Uppsala Software Factory; Kleywegt, G. M. (1997) J. Mol. Biol. 273,371-376). After an additional round of model building and water pickingusing CNX another complete round of refinement was performed. The finalmodel included the DHODH(Met30-Arg396) protein, the cofactorflavinmononucleotide (FMN), one orotate molecule (ORO), one acetatemolecule (ACT), five sulfate ions (SO₄), one molecule of compound 8(INH) and 218 water molecules (TIP).

The model is well refined and shows very good stereochemical geometry.The refinement process which included data from 19.0-1.8 Å resulted inan R-factor of 18.2% and a free R-factor of 19.6% for the R-form andS-form (statistics are given only for R-form). Except for non-glycineand non-proline residues 91.6% of the residues are located in the mostfavoured region of the ramachandran plot and 8.4% cluster in theadditional allowed regions. There are no residues in the disallowedregion. Table 20 summarizes the refinement statistics for compound 8 incomplex with human DHODH. Values in parentheses give the R-factor andR_(free)-factors, respectively, for the last resolution bin ranging from1.91 to 1.8. TABLE 20 Refinement Statistics for DHODH/compound 8 complexR-factor (%) 18.2 (22.1) R_(free) 19.6 (24.6) RMS deviation from idealvalues bond length (Å) 0.005 Bond angle (°) 1.2 Dihedral angles (°) 21.2Improper angles (°) 0.83Structure Determination and Refinement of DHODH/Compound 9 Complex

The structure for the human DHODH(Met30-Arg396) in complex with compound9 was solved using the method of molecular replacement (MR). The freeaccessible pdb entry 1D3G.pdb was used as a search model. The ligandsbrequinar and DDQ as well as all of the water molecules were removedprior to the MR search. The search model included the polypeptide chainof hDHODH(Met30-Arg396), one molecule of orotate, one molecule of thecofactor flavinmononucleotide (FMN) and one acetate molecule which waspresent under the crystallization conditions. A standard rotational andtranslational molecular replacement search at 3.0 Å was performed usingthe program molrep (Collaborative Computational Project, Number 4(1994). Acta Cryst. D50, 760-763.). Solutions for both the rotationaland translational search were well above the next ranking solutions. TheMR resulted in an R-factor of 32.8% and a correlation coefficient of73.6 for the DHODH/compound 9 complex.

In a first round of refinement the MR model was subjected to rigid bodyrefinement and a slow cooling simulated annealing protocol using amaximum likelihood target to remove model bias (Accelrys Inc. CNXprogram suite, CNX2002). Additionally, an individual b-factor refinementwas carried out using standard CNX-protocols. Finally SIGMAA weighted2Fo-Fc and Fo-Fc electron density maps were calculated and displayedtogether with the protein model in the program O (DatOno A B; Jones, T.A., Zou, J. Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,110-119.). The resulting experimental electron density was so excellentthat the conformation of the inhibitor compound 9 could be interpretedunambiguously. The electron density around the five-membered ringcarrying the carboxy group clearly showed the carboxy group in contactwith residues Gln 47 and Arg 136 and a conserved water molecule in aunique brequinar-like conformation addressing subsite 2 only. In thisconformation the sulfur atom of the five membered ring comes into closecontact to Val 134 and Val 143 which form in part subsite 4 (remotehydrophobic pocket).

A pdb file for compound 9 was created using the program MOE (ChemicalComputing Group Inc., MOE 2002.02). After energy minimization thecompound was built into the electron density manually. Topology andparameter files for compound 9 were created using the program Xplo2d(Uppsala Software Factory; Kleywegt, G. M. (1997) J. Mol. Biol. 273,371-376). After an additional round of model building and water pickingusing CNX another complete round of refinement was performed. The finalmodel included the DHODH(Met30-Arg396) protein, the cofactorflavinmononucleotide (FMN), one orotate molecule (ORO), one acetatemolecule (ACT), five sulfate ions (SO₄), one molecule of compound 9(INH) and 291 water molecules (TIP).

The model is well refined and shows very good stereochemical geometry.The refinement process which included data from 17.2-2.0 Å resulted inan R-factor of 18.1% and a free R-factor of 20.0%. Except fornon-glycine and non-proline residues 92.1% of the residues are locatedin the most favoured region of the ramachandran plot and 7.9% cluster inthe additional allowed regions. There are no residues in the disallowedregion. Table 21 summarizes the refinement statistics for compound 9 incomplex with human DHODH. Values in parentheses give the R-factor andR_(free)-factors, respectively, for the last resolution bin ranging from2.13 to 2.0. TABLE 21 Refinement Statistics for DHODH/compound 9 complexR-factor (%) 18.1 (19.7) R_(free) 20.0 (22.0) RMS deviation from idealvalues bond length (Å) 0.005 Bond angle (°) 1.2 Dihedral angles (°) 21.2Improper angles (°) 0.80Structure Determination and Refinement of DHODH/Compound 10 Complex

The structure for the human DHODH(Met30-Arg396) in complex with compound10 was solved using the method of molecular replacement (MR). The freeaccessible pdb entry 1D3G.pdb was used as a search model. The ligandsbrequinar and DDQ as well as all of the water molecules were removedprior to the MR search. The search model included the polypeptide chainof hDHODH(Met30-Arg396), one molecule of orotate, one molecule of thecofactor flavinmononucleotide (FMN) and one acetate molecule which waspresent under the crystallization conditions. A standard rotational andtranslational molecular replacement search at 3.0 Å was performed usingthe program molrep (Collaborative Computational Project, Number 4(1994). Acta Cryst. D50, 760-763.). Solutions for both the rotationaland translational search were well above the next ranking solutions. TheMR resulted in an R-factor of 32.8% and a correlation coefficient of74.1 for the DHODH/compound 10 complex.

In a first round of refinement the MR model was subjected to rigid bodyrefinement and a slow cooling simulated annealing protocol using amaximum likelihood target to remove model bias (Accelrys Inc. CNXprogram suite, CNX2002). Additionally, an individual b-factor refinementwas carried out using standard CNX-protocols. Finally SIGMAA weighted2Fo-Fc and Fo-Fc electron density maps were calculated and displayedtogether with the protein model in the program O (DatOno A B; Jones, T.A., Zou, J. Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,110-119.). The resulting experimental electron density was so excellentthat the conformation of the inhibitor compound 10 could be interpretedunambiguously. The electron density around the five-membered ringcarrying the carboxy group clearly showed the presence of twoalternative conformations of compound 10. In one conformation(conformation A) the carboxy group interacts with residues Gln 47 andArg 136, whereas in the second conformation (conformation B) theinteraction involves residues His 56 and Tyr 356 (see above). For eachconformation a separate DHODH/compound 10 complex was subjected torefinement.

The pdb files for the compound 10 in conformation A and B were createdusing the program MOE (Chemical Computing Group Inc., MOE 2002.02). Bothcompounds were energy minimized and built into the electron densitymanually. Topology and parameter files for compound 10 were createdusing the program Xplo2d (Uppsala Software Factory; Kleywegt, G. M.(1997) J. Mol. Biol., 273, 371-376). After an additional round of modelbuilding and water picking using CNX, another complete round ofrefinement was performed. The final model included the humanDHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide (FMN),one orotate molecule (ORO), two acetate molecules (ACT), four sulfateions (SO₄), one molecule of compound 10 (INH) either in conformation Aor conformation B and 226 water molecules (TIP). Residues which aremissing the coordinate file due to very poor electron density are listedin the header of the pdb files.

The models are well refined and show very good geometry. The refinementprocess which included data from 19.5-1.8 Å resulted in an R-factor of19.5% and a free R-factor of 20.5% for the complex in conformation A andfor the complex in conformation B, respectively. The identical R-factorsindicate that non of the conformers A and B represent a preferredconformation. Except for non-glycine and non-proline residues 91.6% arelocated in the most favoured region of the ramachandran plot and 8.4%cluster in the additional allowed regions. There are no residues in thedisallowed region. Table 22 summarizes the refinement statistics forcompound 10 in complex with human DHODH. Values in parentheses give theR-factor and R_(free)-factors, respectively, for the last resolution binranging from 1.91 to 1.8. TABLE 22 Refinement Statistics forDHODH/compound 10 complex conformation A & B R-factor (%) 19.5 (20.5)R_(free) 20.5 (22.7) RMS deviation from ideal values bond length (Å)0.005 Bond angle (°) 1.2 Dihedral angles (°) 21.9 Improper angles (°)0.82

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

The following compounds are preferred:3-(Biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylic acid;3-(2′-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylicacid;3-(3′-Ethoxy-3,5-difluoro-biphenyl-4-yl-carbamoyl)-thiophene-2-carboxylicacid;3-(3,5-Difluoro-2′,4′-dimethoxy-biphenyl-4-yl-carbamoyl)-thiophene-2-carboxylicacid;3-(2,3,5,6-Tetrafluoro-2′-methoxy-biphenyl-4-yl-carbamoyl)-thiophene-2-carboxylicacid;3-(2′-Chloro-3,5-difluoro-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylicacid; 3-(3,5,2′-Trifluoro-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylicacid;3-(2-Chloro-2′-methoxy-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylicacid;3-(2,3,5,6-Tetrafluoro-3′-trifluoromethoxy-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylicacid;3-(3-Fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylicacid;3-(3,5-Difluoro-3′-trifluoromethoxy-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylicacid; 3-(Biphenyl-4-ylcarbamoyl)-furan-2-carboxylic acid;4-(Biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylic acid;4-(2-Chloro-2′-methoxy-biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylicacid; 4-(3,5,2′-Trifluoro-biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylicacid;4-(3′-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylicacid;4-(2′-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylicacid;4-(3,5-Difluoro-3′-trifluoromethoxy-biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylicacid;4-(3-Fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylicacid; 4-(Biphenyl-4-ylcarbamoyl)-furan-3-carboxylic acid;2-(Biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylic acid;2-(Bi-phenyl-4-ylcarbamoyl)-furan-3-carboxylic acid;3-(3-Fluoro-3′-methoxy-biphenyl-4-yl-carbamoyl)-cyclopent-2-ene-1,2-dicarboxylicacid;2-(3-Fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl)-cyclopent-1-ene-1,3-dicarboxylicacid;2-(3-Fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl)-cyclopent-1-enecarboxylicacid methyl ester; Cyclopent-1-ene-1,2-dicarboxylic acid1-[(3-fluoro-3′-methoxy-biphenyl-4-yl)-amide]2-hydroxyamide;3-Hydroxy-2-(2,3,5,6-tetrafluoro-3′-trifluoromethoxy-biphenyl-4-ylcarbamoyl)-cyclopent-1-enecarboxylicacid;5-Hydroxy-2-(2,3,5,6-tetrafluoro-3′-trifluoromethoxy-biphenyl-4-ylcarbamoyl)-cyclopent-1-enecarboxylicacid;2-(3′-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-3-hydroxy-cyclopent-1-enecarboxylicacid;2-(3′-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-5-hydroxy-cyclo-pent-1-enecarboxylicacid; 2-(1′,3′di-methoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-3-hydroxy-cyclopent-1-enecarboxylicacid;2-(1′,3′di-methoxy-3,5-difluoro-biphenyl-4-yl-carbamoyl)-5-hydroxy-cyclopent-1-enecarboxylicacid;3-Hydroxy-2-(3,5,2′-trifluoro-biphenyl-4-ylcarbamoyl)-cyclopent-1-enecarboxylicacid;5-Hydroxy-2-(3,5,2′-trifluoro-biphenyl-4-ylcarbamoyl)-cyclopent-1-enecarboxylicacid;2-(2-Chloro-2′-methoxy-biphenyl-4-ylcarbamoyl)-3-hydroxy-cyclopent-1-enecarboxylicacid;2-(2-Chloro-2′-methoxy-biphenyl-4-ylcarbamoyl)-5-hydroxy-cyclopent-1-enecarboxylicacid;2-(2′-Chloro-3,5-difluoro-biphenyl-4-ylcarbamoyl)-3-hydroxy-cyclopent-1-enecarboxylicacid;2-(2′-Chloro-3,5-difluoro-biphenyl-4-ylcarbamoyl)-5-hydroxy-cyclopent-1-enecarboxylicacid;2-(3-Fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl)-3-hydroxy-cyclopent-1-enecarboxylicacid;2-(3-Fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl)-5-hydroxy-cyclopent-1-enecarboxylicacid; trans 2-(3-Fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl)-cyclopentanecarboxylic acid;cis-2-(3-Fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl)-cyclopentanecarboxylic acid;2-(2′-Chloro-3,5-difluoro-biphenyl-4-ylcarbamoyl)-cyclopentanecarboxylic acid;2-(3,5-Difluoro-2′,4′-dimethoxy-biphenyl-4-ylcarbamoyl)-cyclopentanecarboxylic acid;2-(3′-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-cyclopentanecarboxylic acid;2-(2′-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-cyclopentanecarboxylic acid; 2-(Biphenyl-4-ylcarbamoyl)-cyclopentane carboxylicacid;2-(2,3,5,6-Tetrafluoro-3′-trifluoro-methoxy-biphenyl-4-ylcarbamoyl)-cyclopentanecarboxylic acid;2-(3,5-Difluoro-3′-trifluoro-methoxy-biphenyl-4-yl-carbamoyl)-cyclopentanecarboxylic acid LENGTHY TABLE REFERENCED HEREUS20070027193A1-20070201-T00001 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027193A1-20070201-T00002 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027193A1-20070201-T00003 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027193A1-20070201-T00004 Please refer to the end of thespecification for access instructions. LENGTHY TABLE The patentapplication contains a lengthy table section. A copy of the table isavailable in electronic form from the USPTO web site(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070027193A1)An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. A compound capable of binding to the ubiquinone binding site of DHODH which contains an aromatic or non-aromatic ring system as a core structure, a group capable of forming a hydrogen bond and/or interacting ionically with structural elements of subsite 2 or 3 of the ubiquinone binding site of DHODH and a group capable of interacting hydrophobically with structural elements of subsite 1 of the ubiquinone binding site of DHODH with the proviso that the following compounds are excluded: compounds of the general formula

wherein A is a non-aromatic ring system containing five carbon atoms, wherein the ring system comprises at least one double bond and wherein one or more of the carbon atoms in the ring can be replaced by a group X, wherein X is selected from the group consisting of S, O, N, NR⁴, SO or SO₂, and wherein one or more of the carbon atoms of the ring can carry a substituent R¹; D is O, S, SO₂, NR⁴, or CH₂; Z¹ and Z² are independent from each other O, S, or NR⁵; R¹ is independently H, halogen, haloalkyl, haloalkyloxy or alkyl; R² is H, OR⁶, or NHR⁷; R³ is H, alkyl, cycloalkyl, aryl, arylalkyl, alkoxy, O-aryl; O-cycloalkyl, halogen, aminoalkyl, alkylamino, hydroxylamino, hydroxylalkyl, haloalkyl, haloalkyloxy, heteroaryl, alkylthio, S-aryl, or S-cycloalkyl; R⁴ is H, alkyl, cycloalkyl, aryl, or heteroaryl; R⁵ is H, OH, alkoxy, O-aryl, alkyl, or aryl; R⁶ is H, alkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, alkylaryl, alkoxyalkyl, acylmethyl, (acyloxy)alkyl, non-symmetrical (acyloxy)alkyldiester, or dialkylphosphate; R⁷ is H, alkyl, aryl, alkoxy, O-aryl, cycloalkyl, or O-cycloalkyl; R⁸ is hydrogen or alkyl; E is an alkyl or cycloalkyl group or a monocyclic or polycyclic substituted or unsubstituted ring system which may contain one or more groups X and which contains at least one aromatic ring; Y is hydrogen, halogen, haloalkyl, haloalkyloxy, alkyl, cycloalkyl, a monocyclic or polycyclic substituted or unsubstituted ring system which may contain one or more groups X and which contains at least one aromatic ring or

m is 0 or 1; n is 0 or 1; p is 0 or 1; r is 0 or 1; and q is 0 to 10; and compounds of the general formula

wherein A is a non-aromatic ring system containing 4, 6, 7 or 8 carbon atoms, wherein the ring system comprises at least one double bond and wherein one or more of the carbon atoms in the ring can be replaced by a group X, wherein X is selected from the group consisting of S, O, N, NR⁴, SO or SO₂, and wherein one or more of the carbon atoms of the ring can carry a substituent R¹; D, Z¹, Z², R¹, R³, R⁴, R⁵, R⁶, R⁸, and E are as defined above, R² is H, or OR⁶; Y is a monocyclic or polycyclic substituted or unsubstituted ring system which may contain one or more groups X and which contains at least one aromatic ring or

m, n, p, r, and q are as defined above.
 2. The compound of claim 1 wherein the non-aromatic ring system is a monocyclic ring.
 3. The compound of claim 1 wherein the non-aromatic ring system is a 5-membered ring.
 4. The compound of claim 1 wherein the 5-membered ring is a cyclopentene ring.
 5. The compound of claim 1 wherein the group connecting the core with the hydrophobic group is bonded to the carbon atom participating in the double bond of the cyclopentene ring.
 6. The compound of claim 1 wherein the non-aromatic ring system is an optionally substituted ring system containing 4 to 8 carbon atoms, wherein the ring system comprises at least one double bond and wherein one or more of the carbon atoms in the ring may be substituted by a group X, wherein X is selected from the group consisting of S, O, N, NH, NHR, SO or SO₂ and R is an alkyl group or an unsaturated or saturated carbocycle.
 7. The compound of claim 1 wherein the non-aromatic ring system is a condensed ring system comprising a 5-membered non-aromatic ring and a 6-membered aromatic or non-aromatic ring.
 8. The compound of claim 7 wherein the 6-membered ring contains one or two nitrogen atoms as heteroatom(s).
 9. The compound of claim 8 wherein the group connecting the core with the hydrophobic group is bonded to said nitrogen heteroatom or one of said nitrogen atoms.
 10. The compound of claim 1 wherein the group capable of forming a hydrogen bond and/or interacting ionically with structural elements of subsite 2 or 3 is capable of binding alternatively to said subsite 2 or subsite
 3. 11. The compound of claim 1 containing an additional group capable of forming a hydrogen bond and/or ionically interacting with structural elements of subsite 2 or
 3. 12. The compound of claim 1 containing an additional group capable of forming a hydrogen bond and/or ionically interacting with structural elements of subsite 2 or 3 and wherein one group is capable of interacting with subsite 2 and the other group is capable of interacting with subsite
 3. 13. The compound of claim 1 wherein the group capable of forming a hydrogen bond and/or interacting ionically with structural elements of subsite 2 or 3 is capable of binding to said subsite 3 only.
 14. A compound capable of binding to the ubiquinone binding site of DHODH which contains a ring system as a core structure, a group capable of forming a hydrogen bond and/or interacting ionically with residues His 56 and/or Tyr 356 of subsite 3 of the ubiquinone binding site of DHODH and a group capable of interacting hydrophobically with structural elements of subsite 1 of the ubiquinone binding site of DHODH.
 15. The compound of claim 14 wherein the group capable of interacting with subsite 3 of DHODH forms a hydrogen bond with residue Tyr 147 of subsite 3 of DHODH.
 16. The compound of claim 14 wherein the group capable of forming a hydrogen bond and/or interacting ionically is capable of binding to said subsite 3 only.
 17. The compound of claim 14 additionally containing a group capable of forming a hydrogen bond and/or interacting ionically with subsite 2 of the ubiquinone binding site of DHODH.
 18. The compound of claim 14 wherein the ring system is an aromatic ring system.
 19. The compound of claim 1 which is crystallizable with DHODH.
 20. The compound of claim 1 wherein the group connecting the core with the hydrophobic group is selected from —NH—, —O—, —CO—, —NHCONH—, —NHCO— and —CONH—.
 21. The compound of claim 1 wherein the group capable of interacting with subsite 2 and/or 3 of DHODH is at least one group selected from the group consisting of —SO₃H, —OH, —NO₂, —CN, CF₃, ═O, and —COOH.
 22. The compound of claim 1 wherein the group capable of interacting with subsite 2 or 3 of DHODH is an anion.
 23. The compound of claim 1 wherein the group capable of interacting with subsite 2 or 3 of DHODH is a carboxylic group.
 24. The compound of claim 1 wherein the group capable of interacting with subsite 2 or 3 of DHODH is an anion which interacts with residues Gln 47 and/or Arg 136 of subsite 2 of DHODH.
 25. The compound of claim the group capable of interacting with subsite 2 or 3 of DHODH is a carboxylic group which is a substituent of the ring system.
 26. The compound of claim 1 wherein the hydrophobic group is capable of interacting with the hydrophobic pocket of subsite 1 of DHODH comprising amino acid residues Leu 142, Met 43, Leu 46, Ala 55, Ala 59, Phe 98, Met 111, Leu 359, and Pro
 364. 27. The compound of claim 1 wherein the hydrophobic group is selected from optionally substituted monocyclic or bicyclic aryl groups.
 28. The compound of claim 1 wherein the hydrophobic group is an optionally substituted biphenyl group.
 29. The compound of claim 1 wherein the hydrophobic group is an optionally substituted benzyl phenyl ether group.
 30. The compound of claim 1 wherein the hydrophobic group has at least one substituent selected from the group consisting of F, Cl, Br, I, CF₃, OCF₃, and OCH₃.
 31. The compound of claim 1 wherein the DHODH is human DHODH consisting of amino acids Met30 to Arg396.
 32. The compound of claim 1 having an IC50 value in the DHODH activity test of less than 500 nM.
 33. The compound of claim 1 having an IC50 value of less than 300 nM.
 34. The compound of claim 1 having an IC50 value of less than 100 nM.
 35. A compound of claim 1 which inhibits the proliferation of human PBMC's by more than 50% with and IC50 of less than 100 μM.
 36. The compound of claim 1 which inhibits the proliferation of human PBMC's by more than 50% with and IC50 of less than 50 μM
 37. The compound of claim 1 which inhibits the proliferation of human PBMC's by more than 50% with and IC50 of less than 10 μM.
 38. The compound of claim 1 which inhibits the proliferation of human PBMC's by more than 50% with and IC50 of less than 5 μM.
 39. The compound of claim 1 which is a compound of the general formula (I)

or salts or isomeres thereof, wherein A is a 4-8 membered non-aromatic ring system, wherein one or more of the carbon atoms in the ring can be replaced by a group X, wherein X is selected from the group consisting of S, O, N, NR⁴, SO, CO or SO₂; D is O, S, SO₂, NR⁴ or CH₂; Z¹ and Z² are independent from each other O, S, or NR⁵; R¹ independently represents H, halogen, haloalkyl, haloalkyloxy —CO₂R″, —SO₃H, —OH, —CONR*R″, —CR″O, —SO₂—NR*R″, —NO₂, —SO₂—R″, —SO—R*, —CN, alkoxy, alkylthio, aryl, —NR″—CO₂—R′, —NR″—CO—R*, —NR″—SO₂—R′, —O—CO—R*, —NR*R″, —NR*OR″—O—CO₂—R*, —O—CO—NR*R″; cycloalkyl, alkylamino, hydroxyalkylamino, —SH, heteroaryl, or alkyl; R* independently represents H, alkyl, cycloalkyl, aminoalkyl, alkoxy, —OH, —SH, alkylthio, hydroxyalkyl, haloalkyl, haloalkyloxy, aryl or heteroaryl; R′ independently represents H, —CO₂R″, —CONHR″, —CR″O, —SO₂NR″, —NR″—CO-haloalkyl, —NO₂, —NR″—SO₂-haloalkyl, —NR″—SO₂-alkyl, —SO₂-alkyl, —NR″—CO-alkyl, —CN, alkyl, cycloalkyl, aminoalkyl, alkylamino, alkoxy, —OH, —SH, —NR″R*, —NR″OR*, alkylthio, hydroxyalkyl, hydroxyalkylamino, halogen, haloalkyl, haloalkyloxy, aryl, arylalkyl or heteroaryl; R″ independently represents hydrogen, haloalkyl, hydroxyalkyl, alkyl, cycloalkyl, aryl, heteroaryl or aminoalkyl; R² is H, OR⁶, NHR⁷, or R² togehter with the nitrogen atom to which R⁸ is attached forms a 5 or 6 membered heterocyclic ring with the proviso that R² is —[CH₂]₀₋₃ and R⁸ is absent; R³ is H, alkyl, cycloalkyl, aryl, arylalkyl, alkoxy, O-aryl; O-cycloalkyl, halogen, aminoalkyl, alkylamino, hydroxylamino, hydroxylalkyl, haloalkyl, haloalkyloxy, heteroaryl, alkylthio, S-aryl, or S-cycloalkyl; R⁴ is H, alkyl, cycloalkyl, aryl, or heteroaryl; R⁵ is H, OH, alkoxy, O-aryl, alkyl, or aryl; R⁶ is H, alkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, alkylaryl, alkoxyalkyl, acylmethyl, (acyloxy)alkyl, non-symmetrical (acyloxy)alkyldiester, or dialkylphosphate; R⁷ is H, alkyl, aryl, alkoxy, O-aryl, cycloalkyl, or O-cycloalkyl; R⁸ is hydrogen or alkyl; E is an alkyl or cycloalkyl group or a monocyclic or polycyclic substituted or unsubstituted ring system which may contain one or more groups X and which contains at least one aromatic ring; Y is hydrogen, halogen, haloalkyl, haloalkyloxy, alkyl, cycloalkyl, a monocyclic or polycyclic substituted or unsubstituted ring system which may contain one or more groups X and which contains at least one aromatic ring or

m is 0 or 1; n is 0 or 1; p is 0 or 1; q is 0 or 1; t is 1 to 3; with the proviso that trans-2-[4-(Naphthalin-2-yl)thiazol-2-ylaminocarbonyl]cyclopentane carboxylic acid is excluded.
 40. The compound of claim 1 which is a compound of the general formula (II)

or salts or isomeres thereof, wherein A is a 3-8 membered non-aromatic ring system, wherein the ring system comprises at least one double bond and wherein one or more of the carbon atoms in the ring can be replaced by a group X, wherein X is selected from the group consisting of S, O, N, NR⁴, SO, CO or SO₂, wherein, when r=0, there is no double bond between the carbon atoms carrying the substituents —CZ¹- and —CZ²-; D is O, S, SO₂, NR⁴ or CH₂; Z¹ and Z² are independent from each other O, S, or NR⁵; R¹ independently represents H, halogen, haloalkyl, haloalkyloxy —CO₂R″, —SO₃H, —OH, —CONR*R″, —CR″O, —SO₂—NR*R″, —NO₂, —SO₂—R″, —SO—R*, —CN, alkoxy, alkylthio, aryl, —NR —CO₂—R′, —NR′—CO—R*, —NR —SO₂—R′, —O—CO—R*, —NR*R″, —NR*OR″—O—CO₂—R*, —O—CO—NR*R″; cycloalkyl, alkylamino, hydroxyalkylamino, —SH, heteroaryl, or alkyl; R* independently represents H, alkyl, cycloalkyl, aminoalkyl, alkoxy, —OH, —SH, alkylthio, hydroxyalkyl, haloalkyl, haloalkyloxy, aryl or heteroaryl; R′ independently represents H, —CO₂R″, —CONHR″, —CR″O, —SO₂NR″, —NR″—CO-haloalkyl, —NO₂, —NR″—SO₂-haloalkyl, —NR″—SO₂-alkyl, —SO₂-alkyl, —NR″—CO-alkyl, —CN, alkyl, cycloalkyl, aminoalkyl, alkylamino, alkoxy, —OH, —SH, —NR″R*, —NR″OR*, alkylthio, hydroxyalkyl, hydroxyalkylamino, halogen, haloalkyl, haloalkyloxy, aryl, arylalkyl or heteroaryl; R″ independently represents hydrogen, haloalkyl, hydroxyalkyl, alkyl, cycloalkyl, aryl, heteroaryl or aminoalkyl; R² is H, OR⁶, NHR⁷, NHOR⁶ or R² togehter with the nitrogen atom to which R⁸ is attached forms a 5 or 6 membered heterocyclic ring with the proviso that R² is —[CH₂]₀₋₃ and R⁸ is absent; R³ is H, alkyl, cycloalkyl, aryl, arylalkyl, alkoxy, O-aryl; O-cycloalkyl, halogen, aminoalkyl, alkylamino, hydroxylamino, hydroxylalkyl, haloalkyl, haloalkyloxy, heteroaryl, alkylthio, S-aryl, or S-cycloalkyl; R⁴ is H, alkyl, cycloalkyl, aryl, or heteroaryl; R⁵ is H, OH, alkoxy, O-aryl, alkyl, or aryl; R⁶ is H, alkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, alkylaryl, alkoxyalkyl, acylmethyl, (acyloxy)alkyl, non-symmetrical (acyloxy)alkyldiester, or dialkylphosphate; R⁷ is H, alkyl, aryl, alkoxy, O-aryl, cycloalkyl, or O-cycloalkyl; R⁸ is hydrogen or alkyl; E is an alkyl or cycloalkyl group or a monocyclic or polycyclic substituted or unsubstituted ring system which may contain one or more groups X and which contains at least one aromatic ring; Y is hydrogen, halogen, haloalkyl, haloalkyloxy, alkyl, cycloalkyl, a monocyclic or polycyclic substituted or unsubstituted ring system which may contain one or more groups X and which contains at least one aromatic ring or

m is 0 or 1; n is 0 or 1; p is 0 or 1; r is 0 or 1; and q is 0 or 1; t is 1 to 3;
 41. The compound according to claim 39, wherein R¹ is selected from the group consisting of OH, CO₂H and SO₃H.
 42. The compound according to claim 39, wherein both Z¹ and Z² are O.
 43. The compound according to claim 39, wherein E is selected from the group consisting of phenyl, 1-naphtyl, 2-naphthyl, 1-anthracyl and 2-anthracyl and is optionally substituted with one or more substituents R′.
 44. The compound according to claim 39, wherein q=0, t=1, A is a carbocyclic non-aromatic ring system, Y is H or F, and E is phenyl which is optionally substituted with at least one substituent selected from the group consisting of Cl, F, CF₃, OCF₃, O-methyl and O-ethyl.
 45. The compound according to claim 39, wherein q=0, t=1, A is a carbocyclic non-aromatic ring system, and E and Y are phenylene and phenyl, respectively, wherein E is optionally substituted with at least one substituent selected from the group consisting of Cl and F and Y is optionally substituted with at least one substituent selected from the group consisting of O-methyl, O-ethyl, OCF₃, Cl and F.
 46. The compound according to claim 39, wherein Y and —NCR⁸ are in para position on E.
 47. A compound of the general formula (II) and salts and physiologically functional derivatives thereof,

wherein A is a heteroaromatic 5-membered ring system containing one or more groups X selected from the group consisting of S, O, N, NR⁴, SO₂ and SO; D is O, S, SO₂, NR⁴, or CH₂; Z¹ and Z² are independent from each other O, S, or NR⁵; R¹ independently represents H, halogen, haloalkyl, haloalkyloxy —CO₂R″, —SO₃H, —OH, —CONR*R , —CR″O, —SO₂—NR*R″, —NO₂, —SO₂—R″, —SO—R*, —CN, alkoxy, alkylthio, aryl, —NR″—CO₂—R′, —NR″—CO—R*, —NR″—SO₂—R′, —O—CO—R*, —O—CO₂—R*, —O—CO—NR*R″; cycloalkyl, alkylamino, hydroxyalkylamino, —SH, heteroaryl, or alkyl; R* independently represents H, alkyl, cycloalkyl, aminoalkyl, alkoxy, —OH, —SH, alkylthio, hydroxyalkyl, haloalkyl, haloalkyloxy, aryl or heteroaryl; R′ independently represents H, —CO₂R″, —CONHR″, —CR″O, —SO₂NR″, —NR″—CO-haloalkyl, —NO₂, —NR″—SO₂-haloalkyl, —NR″—SO₂-alkyl, —SO₂-alkyl, —NR″—CO-alkyl, —CN, alkyl, cycloalkyl, aminoalkyl, alkylamino, alkoxy, —OH, —SH, alkylthio, hydroxyalkyl, hydroxyalkylamino, halogen, haloalkyl, haloalkyloxy, aryl, arylalkyl or heteroaryl; R″ independently represents hydrogen, haloalkyl, hydroxyalkyl, alkyl, cycloalkyl, aryl, heteroaryl or aminoalkyl; R² is H or OR¹, NHR⁷, NR⁷OR or R² togehter with the nitrogen atom which is attached to R⁸ form a 5 or 6 membered heteroyclic ring with the proviso that R² is -[CH₂], and R⁸ is absent; R³ is H, alkyl, cycloalkyl, aryl, alkoxy, O-aryl; O-cycloalkyl, halogen, aminoalkyl, alkylamino, hydroxylamino, hydroxylalkyl, haloalkyloxy, heteroaryl, alkylthio, S-aryl; S-cycloalkyl, arylalkyl, or haloalkyl; R⁴ is H, alkyl, cycloalkyl, aryl or heteroaryl; R⁵ is H, OH, alkoxy, O-aryl, alkyl or aryl; R⁶ is H, alkyl, cycloalkyl, aryl, arylalkyl, heteroaryl, alkylaryl, alkoxyalkyl, acylmethyl, (acyloxy)alkyl, non-symmetrical (acyloxy)alkyldiester, or dialkylphosphate; R⁷ is H, OH, alkyl, aryl, alkoxy, O-aryl, cycloalkyl, or O-cycloalkyl; R⁸ is hydrogen, or alkyl; E is an alkyl or cycloalkyl group or a monocyclic or polycyclic substituted or unsubstituted ring system which may contain one or more groups X and which contains at least one aromatic ring; Y is hydrogen, halogen, haloalkyl, haloalkyloxy, alkyl, cycloalkyl, a monocyclic or polycyclic substituted or unsubstituted ring system which may contain one or more groups X and which contains at least one aromatic ring or

m is 0 or 1; n is 0 or 1; p is 0 or 1; q is 0 or 1; s is 0 to 2; and t is 0 to 3; with the proviso that the following compounds are excluded: compounds wherein ring A contains five atoms, Z¹=Z²═O, and R² togehter with the nitrogen atom which is attached to R⁸ forms a 5 membered heteroyclic ring with the proviso that R² is -[CH₂]₅, R⁸ is absent and s is 0; compounds wherein ring A contains three carbon atoms and two nitrogen atoms, Z¹=Z²═O, and R² togehter with the nitrogen atom which is attached to R⁸ form a 5 membered heteroyclic ring with the proviso that R² is -[CH₂]₅, R⁸ is absent and s is 0; 4-[4-(naphthalin-2-yl)thiazol-2-ylaminocarbonyl]-furan-3-carboxylic acid; and 5-[4-(naphthalin-2-yl]thiazol-2-ylaminocarbonyl]-2H-[1,2,3]-triazole-4-carboxylic acid.
 48. A crystal containing a polypeptide and a compound of claim 1 wherein the polypeptide includes a ubiquinone binding site of DHODH.
 49. A crystal containing a polypeptide and a compound of claim 39 wherein the polypeptide includes a ubiquinone binding site of DHODH.
 50. A crystal containing a polypeptide and a compound of claim 40 wherein the polypeptide includes a ubiquinone binding site of DHODH.
 51. A crystal containing a polypeptide and a compound of claim 47 wherein the polypeptide includes a ubiquinone binding site of DHODH.
 52. The crystal of claim 48 wherein the compound is compound 1


53. The crystal of claim 48 wherein the compound is compound 2


54. The crystal of claim 48 wherein no salt bridge or hydrogen bridge is formed between the compound and an amino acid residue in subsite 2 of DHODH.
 55. The crystal of claim 48 wherein no salt bridge or hydrogen bridge is formed between the carboxylic group of Compound 1 or Compound 2 and the sidechain of Arg136.
 56. The crystal of claim 48 wherein the compound forms a hydrogen bond and/or interacts ionically with structural elements of subsite 3 only.
 57. The crystal of claim 48 wherein the compound forms hydrogen bond(s) with residues His 56 and/or Tyr 356 of subsite 3 of DHODH.
 58. The crystal of claim 48 wherein the compound forms a hydrogen bond with residue Tyr 147 of subsite 3 of DHODH.
 59. A method of identifying a compound which is an inhibitor of DHODH comprising the steps of (a) obtaining the atomic coordinates of the crystal of claim 48; (b) using said atomic coordinates to define the ubiquinone binding site of DHODH; and (c) identifying a compound which fits the ubiquinone binding site of DHODH.
 60. A method of identifying a compound which is an inhibitor of DHODH comprising the steps of (d) obtaining the atomic coordinates of the crystal of claim 49; (e) using said atomic coordinates to define the ubiquinone binding site of DHODH; and (f) identifying a compound which fits the ubiquinone binding site of DHODH.
 61. A method of identifying a compound which is an inhibitor of DHODH comprising the steps of (g) obtaining the atomic coordinates of the crystal of claim 50; (h) using said atomic coordinates to define the ubiquinone binding site of DHODH; and (i) identifying a compound which fits the ubiquinone binding site of DHODH.
 62. A method of identifying a compound which is an inhibitor of DHODH comprising the steps of (j) obtaining the atomic coordinates of the crystal of claim 51; (k) using said atomic coordinates to define the ubiquinone binding site of DHODH; and (l) identifying a compound which fits the ubiquinone binding site of DHODH.
 63. A method of identifying a compound which is an inhibitor of DHODH comprising the steps of (m) obtaining the atomic coordinates of the crystal of claim 48; (n) using said atomic coordinates to define the structural requirements of the inhibitor contained in the polypeptide-inhibitor complex; and (o) designing a compound on the basis of said structural requirements.
 64. A method of identifying a compound which is an inhibitor of DHODH comprising the steps of (p) obtaining the atomic coordinates of the crystal of claim 49; (q) using said atomic coordinates to define the structural requirements of the inhibitor contained in the polypeptide-inhibitor complex; and (r) designing a compound on the basis of said structural requirements.
 65. A method of identifying a compound which is an inhibitor of DHODH comprising the steps of (s) obtaining the atomic coordinates of the crystal of claim 50; (t) using said atomic coordinates to define the structural requirements of the inhibitor contained in the polypeptide-inhibitor complex; and (u) designing a compound on the basis of said structural requirements.
 66. A method of identifying a compound which is an inhibitor of DHODH comprising the steps of (v) obtaining the atomic coordinates of the crystal of claim 51; (w) using said atomic coordinates to define the structural requirements of the inhibitor contained in the polypeptide-inhibitor complex; and (x) designing a compound on the basis of said structural requirements.
 67. A compound obtainable by the method of claim
 59. 68. A compound obtainable by the method of claim
 60. 69. A compound obtainable by the method of claim
 61. 70. A compound obtainable by the method of claim
 62. 71. A compound obtainable by the method of claim
 63. 72. A compound obtainable by the method of claim
 64. 73. A compound obtainable by the method of claim
 65. 74. A compound obtainable by the method of claim
 66. 75. A method for treatment of a disease or a therapeutic indication in which inhibition of dihydrooratate dehydrogenase is beneficial which comprises administering to a mammal an effective amount of a compound according to claim 1, a physiologically functional derivative or a pharmacologically tolerable salt thereof.
 76. A method for treatment of a disease or a therapeutic indication in which inhibition of dihydrooratate dehydrogenase is beneficial which comprises administering to a mammal an effective amount of a compound according to claim 39, a physiologically functional derivative or a pharmacologically tolerable salt thereof.
 77. A method for treatment of a disease or a therapeutic indication in which inhibition of dihydrooratate dehydrogenase is beneficial which comprises administering to a mammal an effective amount of a compound according to claim 40, a physiologically functional derivative or a pharmacologically tolerable salt thereof.
 78. A method for treatment of a disease or a therapeutic indication in which inhibition of dihydrooratate dehydrogenase is beneficial which comprises administering to a mammal an effective amount of a compound according to claim 47, a physiologically functional derivative or a pharmacologically tolerable salt thereof.
 79. A method for treatment of a disease or a therapeutic indication in which inhibition of dihydrooratate dehydrogenase is beneficial which comprises administering to a mammal an effective amount of a compound according to claim 67, a physiologically functional derivative or a pharmacologically tolerable salt thereof.
 80. A method for treatment of a disease or a therapeutic indication in which inhibition of dihydrooratate dehydrogenase is beneficial which comprises administering to a mammal an effective amount of a compound according to claim 68, a physiologically functional derivative or a pharmacologically tolerable salt thereof.
 81. A method for treatment of a disease or a therapeutic indication in which inhibition of dihydrooratate dehydrogenase is beneficial which comprises administering to a mammal an effective amount of a compound according to claim 69, a physiologically functional derivative or a pharmacologically tolerable salt thereof.
 82. A method for treatment of a disease or a therapeutic indication in which inhibition of dihydrooratate dehydrogenase is beneficial which comprises administering to a mammal an effective amount of a compound according to claim 70, a physiologically functional derivative or a pharmacologically tolerable salt thereof.
 83. A method for treatment of a disease or a therapeutic indication in which inhibition of dihydrooratate dehydrogenase is beneficial which comprises administering to a mammal an effective amount of a compound according to claim 71, a physiologically functional derivative or a pharmacologically tolerable salt thereof.
 84. A method for treatment of a disease or a therapeutic indication in which inhibition of dihydrooratate dehydrogenase is beneficial which comprises administering to a mammal an effective amount of a compound according to claim 72, a physiologically functional derivative or a pharmacologically tolerable salt thereof.
 85. A method for treatment of a disease or a therapeutic indication in which inhibition of dihydrooratate dehydrogenase is beneficial which comprises administering to a mammal an effective amount of a compound according to claim 73, a physiologically functional derivative or a pharmacologically tolerable salt thereof.
 86. A method for treatment of a disease or a therapeutic indication in which inhibition of dihydrooratate dehydrogenase is beneficial which comprises administering to a mammal an effective amount of a compound according to claim 74, a physiologically functional derivative or a pharmacologically tolerable salt thereof.
 87. The method of claim 75 wherein the disease or indication is selected from the group consisting of rheumatism, acute immunological disorders, autoimmune diseases, diseases caused by malignant cell proliferation, inflammatory diseases, diseases that are caused by protozoal infestations in humans and animals, diseases that are caused by viral infections and Pneumocystis carinii, fibrosis, uveitis, rhinitis, asthma or athropathy.
 88. The method of claim 76 wherein the disease or indication is selected from the group consisting of rheumatism, acute immunological disorders, autoimmune diseases, diseases caused by malignant cell proliferation, inflammatory diseases, diseases that are caused by protozoal infestations in humans and animals, diseases that are caused by viral infections and Pneumocystis carinii, fibrosis, uveitis, rhinitis, asthma or athropathy.
 89. The method of claim 77 wherein the disease or indication is selected from the group consisting of rheumatism, acute immunological disorders, autoimmune diseases, diseases caused by malignant cell proliferation, inflammatory diseases, diseases that are caused by protozoal infestations in humans and animals, diseases that are caused by viral infections and Pneumocystis carinii, fibrosis, uveitis, rhinitis, asthma or athropathy.
 90. The method of claim 78 wherein the disease or indication is selected from the group consisting of rheumatism, acute immunological disorders, autoimmune diseases, diseases caused by malignant cell proliferation, inflammatory diseases, diseases that are caused by protozoal infestations in humans and animals, diseases that are caused by viral infections and Pneumocystis carinii, fibrosis, uveitis, rhinitis, asthma or athropathy.
 91. The method of claim 79 wherein the disease or indication is selected from the group consisting of rheumatism, acute immunological disorders, autoimmune diseases, diseases caused by malignant cell proliferation, inflammatory diseases, diseases that are caused by protozoal infestations in humans and animals, diseases that are caused by viral infections and Pneumocystis carinii, fibrosis, uveitis, rhinitis, asthma or athropathy.
 92. The method of claim 80 wherein the disease or indication is selected from the group consisting of rheumatism, acute immunological disorders, autoimmune diseases, diseases caused by malignant cell proliferation, inflammatory diseases, diseases that are caused by protozoal infestations in humans and animals, diseases that are caused by viral infections and Pneumocystis carinii, fibrosis, uveitis, rhinitis, asthma or athropathy.
 93. The method of claim 81 wherein the disease or indication is selected from the group consisting of rheumatism, acute immunological disorders, autoimmune diseases, diseases caused by malignant cell proliferation, inflammatory diseases, diseases that are caused by protozoal infestations in humans and animals, diseases that are caused by viral infections and Pneumocystis carinii, fibrosis, uveitis, rhinitis, asthma or athropathy.
 94. The method of claim 82 wherein the disease or indication is selected from the group consisting of rheumatism, acute immunological disorders, autoimmune diseases, diseases caused by malignant cell proliferation, inflammatory diseases, diseases that are caused by protozoal infestations in humans and animals, diseases that are caused by viral infections and Pneumocystis carinii, fibrosis, uveitis, rhinitis, asthma or athropathy.
 95. The method of claim 83 wherein the disease or indication is selected from the group consisting of rheumatism, acute immunological disorders, autoimmune diseases, diseases caused by malignant cell proliferation, inflammatory diseases, diseases that are caused by protozoal infestations in humans and animals, diseases that are caused by viral infections and Pneumocystis carinii, fibrosis, uveitis, rhinitis, asthma or athropathy.
 96. The method of claim 84 wherein the disease or indication is selected from the group consisting of rheumatism, acute immunological disorders, autoimmune diseases, diseases caused by malignant cell proliferation, inflammatory diseases, diseases that are caused by protozoal infestations in humans and animals, diseases that are caused by viral infections and Pneumocystis carinii, fibrosis, uveitis, rhinitis, asthma or athropathy.
 97. The method of claim 85 wherein the disease or indication is selected from the group consisting of rheumatism, acute immunological disorders, autoimmune diseases, diseases caused by malignant cell proliferation, inflammatory diseases, diseases that are caused by protozoal infestations in humans and animals, diseases that are caused by viral infections and Pneumocystis carinii, fibrosis, uveitis, rhinitis, asthma or athropathy.
 98. The method of claim 86 wherein the disease or indication is selected from the group consisting of rheumatism, acute immunological disorders, autoimmune diseases, diseases caused by malignant cell proliferation, inflammatory diseases, diseases that are caused by protozoal infestations in humans and animals, diseases that are caused by viral infections and Pneumocystis carinii, fibrosis, uveitis, rhinitis, asthma or athropathy. 